Measuring Human Cytokine Responses

18 Measuring Human Cytokine Responses

Hans Yssel1, John Wijdenes2, René de Waal Malefyt3, Jean-François Mathieu4 and Jérôme Pène1 1 4

Inserm U844, Montpellier, France; 2 Gen-Probe, Besançon, France; 3 Schering-Plough Biopharma, Palo Alto, CA, USA; BD Biosciences, Erembodegem, Belgium

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CONTENTS

tttttt I. INTRODUCTION The term cytokine has been used to describe a diverse group of low-molecular­ weight (generally <20 kD) protein mediators that have a broad spectrum of immunoregulatory effects and which are produced by a variety of cell types. Although many cytokines were originally defined and named after the particular biological functions that they display, the development of recombinant DNA technology has permitted the classification of a plethora of factors and activities into a growing list of well-defined proteins. From a functional point of view, cytokines have several features in common. • Most, if not all, cytokines have redundant activities, as reflected by their ability to perform similar functions. Among the many examples are interleukin (IL)-2, IL-4, IL-7, IL-10, IL-12, IL-15, IL-21, IL-23, which all have T cell growth-promot­ ing activities, IL-4 and IL-13, which share most of their functional activities, such as induction of B cell proliferation and differentiation, induction of IgG1 and IgE isotype switching, induction of mast cell differentiation and downregulation of secretion of pro-inflammatory cytokines by macrophages, or IL-1, IL-6 and TNF-a, which activate hepatocytes to synthesize acute phase proteins, induce bone marrow epithelium to release neutrophils and act as endogenous pyrogens METHODS IN MICROBIOLOGY, VOLUME 37 0580-9517 DOI 10.1016/S0580-9517(10)37018-8

 2010 Elsevier Ltd. All rights reserved

Measuring Human Cytokine Responses

Introduction Assays for Measuring Cytokine Production Real-Time Quantitative PCR ‘Taqman’ Intracellular Cytokine Staining ELISA ELISPOT Assay BD Cytometric Bead Array (CBA)

on the hypothalamus thereby raising the body temperature, which is believed to help eliminate infections. • Their effects are pleiotropic, affecting many different target cells, which is reflected by the functional expression of receptors for certain cytokines, such as IL-1, IL-2, IL-4, IL-6 or IL-10 on most cells of the immune system. • Their secretion is highly regulated, because of the potential for tissue destruction and other adverse effects, and they often are secreted during bursts of immune responses. • Cytokines interact with specific high-affinity cell surface receptors, which is followed by a cascade of signal transduction events, resulting in mRNA synth­ esis and, eventually, protein secretion. For a large number of IL receptors, these signal transduction events have been shown to be associated with the phosphor­ ylation of specific tyrosine kinases, members of the Jak-Stat signal transduction system (reviewed in Ihle, 2001). In addition, most cytokines are glycosylated, giving rise to considerable molecular heterogeneity, with the different sugar molecules being important for receptor binding and modulation of receptormediated signal transduction. • Many cytokines are able to induce or inhibit each other’s synthesis, including their own, resulting in the creation of regulatory networks. An example of the mutual inducing activity of cytokines, resulting in a positive feedback is the effect of IL-12 and interferon (IFN)-g on each other’s production. Secretion of IL-12 by activated monocytes strongly induces the production of IFN-g by T cells and natural killer (NK) cells, which in turn enhances the production of IL-12. Moreover, at least in the mouse, IFN-g induces a functional IL-12R at the cell surface of T cells, rendering them susceptible to the IFN-g-inducing effect of IL­ 12. The inhibitory action of cytokines is exemplified by IL-4, IL-10 and IL-13, which all efficiently block the secretion of pro-inflammatory cytokines, such as IL-1, IL-6 and TNF-a, by activated macrophages and furthermore enhance the production of IL-1RA by these cells, thus amplifying a negative feedback mechanism. Another example is IL-10, a cytokine with relatively late production kinetics that is able to inhibit its own synthesis via an autocrine feedback mechanism. • Mixtures of cytokines often have synergistic effects, as compared to the effect of each of them separately, resulting in an amplification of responses. For example, the pro-inflammatory cytokines IL-1, IL-6 and TNF-a, which are involved in the so-called acute phase response, synergize to mediate inflammation, shock and even death in response to infectious agents. Cytokines may resemble hormones at first sight, since both are soluble mediators which serve as means of intracellular communication. However, there are impor­ tant differences between the effects of these two families of mediator molecules. Whereas hormones can be easily detected in the circulation, having endocrine (systemic) effects, most cytokines are released locally. Moreover, hormones are released as a result of internal physiologic variation and therefore are important in maintaining a situation of homeostasis. By contrast, cytokines, are produced in short bursts, following external injury, as well as during developmental and effector phases of immune responses, thereby modulating the function of adjacent

440

cells or the cells that produce them. Due to a short half-life time in the circulation, cytokines are generally difficult to detect in serum or plasma, although there are a number of exceptions. For example, during acute inflammation or septic shock, IL-1, IL-6 and TNF-a orchestrate a series of events that induce the acute phase response and they can readily be detected in human serum or plasma. The sys­ temic effects of these cytokines, however, may differ significantly from the effects in the local sphere of influence. IL-1, when released by tissue macrophages, is a co­ stimulatory factor for T cell activation, whereas high systemic levels of IL-1 result in fewer, leukopenia and, as mentioned above, even shock. Cytokines can be classified into a number of categories (listed in Table 1), broadly based on their functional properties, i.e. cytokines: that are involved in innate immunity, that are involved in the regulation of lymphocyte function, that are involved in the regulation of haematopoiesis, that have anti-inflammatory modes of action, that have pro-inflammatory modes of action, that have chemoattractant properties.

As is clear from this list, which is not exhaustive, many cytokines feature in more than one category, which is in line with the notion that redundancy serves the purpose of the immune system to mount an effective and rapid response following inflammation or antigenic challenge. The concomitant release of several cytokines will lead to a rapid mobilization of effector cells at the place of injury or insult to produce the desired biological effects. Cytokines with a broad spectrum of action, such as IL-1 and IL-6, are not only involved in innate immune and inflammatory responses, which form the first line of defence of the host against antigenic chal­ lenge, but also in adaptive immune responses by exerting co-stimulatory effects on T cells and furthermore induce differentiation and growth of B cells. Recently, the interest in the role of cytokines in the induction of inflammatory responses has been raised with the description of a T cell population with a particular cytokine production profile, T helper type 17 (Th17) cells, because of their concomi­ tant production of high levels of IL-17 and IL-22, as well as other pro-imflammatory cytokines including TNF-a, TNF-β and IL-26. The activity of the latter cells is distinct from that of the classical Th1 and Th2 cells, characterized by the production of IFN-g and IL-4, respectively, and although mainly based on the results of experimental animal models, it is currently accepted that activity of the cytokines produced by Th17 cells is strongly associated with excacerbated immune responses that underlie the pathogenesis of various inflammatory diseases such as rheumatoid arthritis, Crohn’s disease or multiple sclerosis (reviewed in Korn et al., 2009). Conversely, in order to prevent exacerbated immune responses, resulting in tissue damage or destruction, many cytokines have downregulatory effects on the production of other cytokines, as well as on their own secretion, in order to dampen the immune response. Examples of such regulatory cytokines are IL-10 and TGF-β, which affect a wide range of target cells. Finally, several cytokines are usually involved in, and required for, the generation of an appropriate immune response and it seems difficult to bestow a more or less prominent role on each of them in view of their interregulatory effects, although there may be a certain 441

Measuring Human Cytokine Responses

• • • • • •

Table 1. Classification of cytokines based on functional properties Cytokine

Biological action

Produced by

1. Cytokines involved in innate immunity IL-1a/β Mediates host response to infectious agents IL-1RA TNF-a IL-6

Natural antagonist of IL-1, blocks IL-1-mediated signal Mediates host response to infectious agents Mediates and regulates inflammatory responses Mediates leucocyte chemotaxis and activation

2. Cytokines involved in regulation of lymphocyte function IL-1 Mediates co-stimulation of T cells Differentiation of Th17 cells IL-2 T cell growth factor, proliferation of B cells IL-4 T cell proliferation, Differentiation of Th2 cells B cell differentiation, IgG4/IgE switching Eosinophil growth and differentiation factor B cell proliferation, enhances Ig secretion in B cells Pre-T and pre-B cell proliferation, differentiation LAK and cytotoxic T cells T cells, NK cells IL-9 Co-stimulator for mast cell-, foetal thymocyte growth IL-10-like cytokines: IL-10, IL-19, IL-20, IL-22, IL-24, IL-26 IL-10 Co-stimulator for T cell, B cell and mastcell growth, downregulation MHC class II/Co-stimulatory molecules on APC IL-19 Unknown IL-20 Keratinocyte proliferation and differentiation IL-22 Stimulates Mono to make TNF-a IL-12 Differentiation of Th1 cells, induction of IFN-g by mono IL-13 B cell differentiation, IgG4/IgE switching IL-5 IL-6 IL-7

IL-15 IL-17 IL-18 IL-21 IL-23 IL-27 TNF-β

T cell, mast cell growth factor Induction of IL-6 and IL-8 production by monocytes induction of CD54 expression by fibroblasts Induction of IFN-g production by T/NK cells Maturation and proliferation of NK cells from BM B cell growth factor, IgG1/IgG3 switching Stimulation of IFN-g production Induction of Th17 cell differentiation/proliferation B cell growth factor, IgG1/IgG3 switching Induction of IL-10 production by Th17 cells Stimulates T cell growth

442

Mø, DC, fibroblasts, astrocytes Mø Mø, T cells Mø, T cells, fibroblasts, chemokines Mø, DC, fibroblasts, astrocytes T cells, NK cells Th2 cells, basophils, mast cells T cells Mø, T cells Bone marrow stromal cells T cells

Mø, T cells, keratinocytes Mø, B cells Keratinocytes, mono T cells, mast cells Mø, B cells T cells, B cells, mastcells Mono Th17 cells Mono T cells, TFH cells, NK cells DC, Mø DC, Mø T cells, B cells

(Continued )

Cytokine

Biological action

Produced by

IFN-g

Mø/NK cell activation, upregulation MHC class I/II on Mono 3. Cytokines involved in the regulation of haemotopoiesis IL-3 Synergistic action in haemotopoiesis IL-5 Growth/differentiation of eosinophils IL-6 Growth/differentiation megakaryocytes G-CSF

Growth/differentiation granulocytic lineage

GM-CSF

Growth/differentiation myelomonocytic lineage

SCF (ckit-L)

Th1 cells, NK cells

T cells, mast cells T cells, mast cells Mono, DC, Mø, T cells Mono, DC, Mø, T cells Mono, DC, Mø, T cells Bone marrow stromal cells Kidney cells

Growth/differentiation haemotopoietic precursor cells Erythropoietin Growth/diff. erythroid progenitor cells 4. Cytokines having anti-inflammatory effects IL-4 Inhibition of pro-inflammatory cytokines by Mono IL-13 Inhibition of pro-inflammatory cytokines by Mono IL-10 Inhibition of pro-inflammatory cytokines by Mono, Inhibition MHC class II expression Mono, inhibition of T cell growth, induction of T regulatory cells TGF-β Inhibition of pro-inflammatory cytokines by Mono Chrondrocytes, Induction of T regulatory cells Mono, DC, Mø, T cells 5. Cytokines having pro-inflammatory effects IL-1 Participates in acute phase response TNF-a Participates in acute phase response IL-6 Participates in acute phase response IL-16 Migratory response in CD4+ T cells/Mono/EO CD8+ T cells TGF-β Induction of Th17 cells with IL-1/IL-6 6. Chemokines (see Zlotnik and Yoshie (2000) for classification of chemokines) DC: Dendritic cells, EO: eosinophils, Mono: monocytes, Mø: macrophages, BM: bone marrow.

hierarchy in the action of cytokines, especially with respect to kinetics of produc­ tion. Therefore, the concomitant inducing and inhibitory effects of many cytokines constitute the basis for the creation of overlapping cytokine networks which are able to tightly regulate an appropriate immune response. Finally, whereas cytokines play a pivotal role in the generation of immune responses, dysregulation of cytokine production has been shown to result in acute of chronic immunopathology. An example is provided by the above-men­ tioned activity of IL-17- and/or IL-22-producing cells that, in the absence of reg­ ulatory mechanisms, will lead to the induction and perpetuation of various inflammatory diseases. Therefore, the possibility to accurately measure cytokine 443

Measuring Human Cytokine Responses

Table 1.

production is not only of great importance for scientific purposes, but also for diagnostic purposes and furthermore is useful for the monitoring of clinical therapies.

tttttt II. ASSAYS FOR MEASURING CYTOKINE PRODUCTION To date, several assays are used to detect and quantify cytokines in serum, body fluids and culture supernatants, as well as their production by cytokine-producing cells. In this chapter we will describe the real-time PCR assay, the enzyme-linked immunosorbent assay (ELISA), ELISPOT assay, the intracellular cytokine staining assay and the BDTM cytometric bead assay. An overview of the advantages and disadvantages of each of these assays, described in this chapter, is shown in Table 2. The detection of cytokines by cell surface staining will be described in Chapter 1 of Volume 32.

Table 2. Advantages and disadvantages of assays for the measurement of cytokines Bioassay Very sensitive Active form of cytokine is detected Can be performed in the absence of standard: activity expressed in arbitrary biological Units Requires use of neutralizing anti-cytokine antibodies to demonstrate specificity Large series of dilutions required to fit results on dose-dependent S-shaped curve Requires labor-intensive tissue culture for relevant responder cells Presence of stimulating agent in culture supernatant may act on responder cells Real time RT-PCR Quantitation of cytokine transcripts

Expensive, technically demanding and calibration difficult

Suitable for high throughput

Detection of cytokine transcripts only and not (secreted) protein

Intracellular staining Specific, but not very sensitive for certain cytokines: sensitivity is dependent on antibody and fixation conditions Requires selection of suitable antibodies that detect chemically fixed cytokines Use in flowcytometry permits analysis of frequency and phenotype of cytokine­ producing cells, as well as kinetics of cytokine production Simultaneous detection of several cytokines produced by same cell Results are not affected by the inducing/inhibitory effects of simultaneously produced cytokines Although theoretically possible, difficult to use under conditions of antigen-specific stimulation Requires viable cells at the end of the stimulation Requires exogenous (recombinant) cytokine to confirm specificity Meaningful interpretation of the results might require kinetics measurements 444

(Continued )

ELISA Specific Results are easy to fit onto dose-dependent S-shaped curve Different modes of activation, including antigen-specific stimulation, can be used Less sensitive than bioassay: sensitivity is dependent on the capture antibody Non-active form of cytokine is also detected Laborious and time consuming when multifunctional analysis is performed Results can be affected by sequestration of cytokines from biological samples by (soluble) cytokine receptors, by inducing/inhibitory effects of simultaneously produced cytokines or by false-positive values with heterophilic antibodies ELISPOT assay Specific, and more sensitive than ELISA, because of local release and capture of cytokines Determination of frequencies of cytokine producing cells although quantification is tedious Simultaneous (although restricted) detection of cytokine production by the same cell Possibility to analyse cytokine production by freshly isolated clinical material without prior in vitro stimulation No possibility to combine flowcytometry and cytokine production Same technical advantages and constraints as ELISA Cytometric Bead Array (CBA) Specific Simultaneous detection and quantification of cytokines by flow cytometry Assay procedure and data processing more rapid as compared to ELISA Only very small sample quantities are required for extensive multifunctional analysis For Bioassay, ELISA, ELISPOT and CBA Accumulation of secreted cytokines is measured: no constraints regarding kinetics of production

A. Activation Procedure Since the production of cytokines is tightly regulated, at the transcriptional, as well as the protein level, cell populations need to be appropriately activated for the required amount of time to allow detection of cytokine transcripts and/or produc­ tion of protein. Activation procedures for each of the cytokine detection assays are nearly identical and will be described first. 1. Protocol

1. Stimulate 106–2.106 ml–1 peripheral blood mononuclear cells (PBMCs) or 106–4.106 ml–1 T cells with either of the following agents (see notes 1 and 2): • Soluble (for PBMCs) or plate-bound (for purified T cells or T cell lines) anti-CD3 monoclonal (m) antibody (Ab) (SPV-T3b, UCHT-L1, OKT3, B-B11 or equivalent) and soluble anti-CD28 mAb (L293, 9.3, BT-3; 1 μg ml–1). Coat plates with 10 μg ml–1 anti-CD3 mAb diluted in phosphate-buffered saline 445

Measuring Human Cytokine Responses

Table 2.

2. 3. 4. 5. 6. 7.

(PBS) and incubate for 18 h at 4°C or for 4 h at 37°C, remove mAb solution, wash twice with PBS and once with culture medium and use in experiment (see note 3). • A combination of mitogenic anti-CD2 mAb (mAb D66 (Rosenthal-Allieri et al., 1995) combined with any sheep red blood cell binding anti-CD2 mAb). • The combination of 12-O-tetradecanoylphorbol-13-acetate (TPA), also called phorbol-12-myristate-13-acetate (PMA) (1 ng ml–1) and the calcium iono­ phore A23187 (500 ng ml–1) (Calbiochem: 524400 and 100105, respectively) or Ionomycin (Calbiochem: 524400), respectively. Use 24-well (final volume 1 ml), 48-well (500 μl) or 96-well (200 μl) plates. When using plate-bound anti-CD3 mAb, spin the culture plates at 100×g for 2 min (see note 3). Incubate the cells at 37°C, 5% CO2. For RT-PCR: harvest the cells after 4–8 h of incubation, spin at 200×g for 10 min at 4°C, carefully remove supernatant and process cells for RNA preparation, as described on page 435. For ELISPOT: gently harvest the cells after 6 h of incubation, transfer to ELI­ SPOT plates and incubate for an additional 20 or 40 h at 37°C, 5% CO2. Analyse filters for presence of cytokines. For ELISA and Cytometric Bead Array (CBA) (see note 4): harvest the supernatants after 24–48 h of incubation, spin at 200×g for 10 min to remove residual cells and analyse for cytokine production. For intracellular staining (see notes 4 and 5): after 4 h of incubation, add 10 μl of Brefeldin A (A stock solution of 10 mg ml–1 Brefeldin A (Epicentre Technologies, Madison, WI: B905MG, or Sigma: B7651) is made in dimethyl sulphoxide (DMSO), diluted 1:10 in PBS, and Brefeldin A is added at a final concentration of 10 μg ml–1 to the cell suspension) and incubate the cells for an additional 2 h at 37°C, 5% CO2. Harvest cells and put on ice, prior to analysis for cytokine production.

2. Notes and recommendations

1. The nature of the cytokine production profile of T cells depends on the mode of activation, and it is therefore recommended to compare different stimulation protocols. Most importantly, these should include activation of the cells with anti-CD3 and anti-CD28 mAbs, which resembles antigen-specific stimulation conditions. Another possibility is the use of superantigens, such as Staphylo­ coccus enterotoxin B (SEB), which bind to certain TCR Vβ gene products. When using PBMC and specific, soluble antigen, use longer incubation periods (up to 4 days) to obtain optimal cytokine production levels. Modes of stimulation invol­ ving polyclonal activators, such as the combination of phorbol ester and calcium ionophore or ionomycine, respectively, should be used only as control activa­ tions to ensure intrinsic capacity of the cells to produce cytokines or as a positive control in analysis of cytokine production in the intracellular staining assay. It is of note that the combination of phorbolester and ionomycine is almost univer­ sially used for the detection of intracellular cytokine production. Although not physiologically comparable to antigen-specific stimulation, this mode of 446

3.

4.

5.

tttttt III. REAL-TIME QUANTITATIVE PCR ‘TAQMAN’ The reverse chain polymerase reaction (RT-PCR) is a powerful technique to analyse gene regulation at the cellular level (Ferre, 1992). However, the advantage of the sensitivity is also a limitation due to the two enzymatic reaction steps. The reverse transcriptase first synthesizes a complementary strand from the RNA with specific, random or oligo dT primers, and this newly synthesized cDNA is subsequently amplified by the Taq polymerase with two specific primers for a sufficient number of times to be analysed. The quantification of a specific cDNA present in the samples is only possible in the exponentiel phase of the enzymatic reaction, where the amount of amplified targets is a linear function of the starting template (Gilland et al., 1990). Real-time quantitative PCR (Higuchi et al., 1992; Higuchi et al., 1993) is a technique that allows for the detection in real time of a PCR product as it accumulates through the successive cycles of a PCR reaction. As a consequence, it is possible to measure product accumulation at the logarithmic amplification phase 447

Measuring Human Cytokine Responses

2.

stimulation indeed results in high frequencies of cytokine-producing cells while maintaining a particular cytokine production profile. Lipopolysaccharide (LPS, 1 µg ml–1; Sigma ref L-2880) is the stimulation of choice for monocytes. To optimaly induce the production of cytokines by these cells, pre-incubate freshly isolated monocytes with 10 ng ml–1 IFN-g for 18 h and stimulate with LPS at a concentration of 100 ng ml–1. Use Iscove’s Modified Dulbecco Medium supplemented with 10% human or foetal calf serum or Yssel’s medium (Yssel et al., 1984), supplemented with 1% human serum (detailed procedure for preparation sent upon request). Spinning cells onto anti-CD3 mAb-coated plates will enhance magnitude of stimulation (H. Yssel, unpublished results). This is especially important for short-time kinetics in RT-PCR assays or for the first incubation of cells for the ELISPOT assay prior to transfer to the filters. Since cytokines are secreted with different kinetics, T cells should be stimulated for at least 24 h (48 h is recommended) before harvesting the supernatants for measurement by ELISA or CBA. When not immediately analysed, culture supernatants should be aliquotted and stored at –80°C. Similarly, cytokine standards should be aliquotted and kept frozen. Repetitive freeze/thaw cycles should be avoided, since most cytokines, in particular IFN-g, will degrade rapidly due to this procedure. For intracellular staining and RT-PCR analysis, 6–8 h of stimulation is optimal for most cytokines. Agents which block intracellular protein transport, such as Brefeldin A and Monensin, have dose- and time-dependent cytotoxic effects and it is not recom­ mended to have either agents included in the cultures for >6 h. Since Monensin has been found to induce the intracellular production of IL-1 and TNF-a in monocytes within 30 min of activation, the use of Brefeldin A, instead of Mon­ ensin, is preferred. Brefeldin A is added at a final concentration of 10 μg ml–1 to the cell suspension. Brefeldin A is toxic: avoid contact with skin, eyes and mucous membranes.

of a PCR reaction, independently of the amount of target sequences present at the onset of the reaction. This has the absolute advantage that quantitation of the PCR product is not performed as an endpoint measurement where availability of reac­ tion components or activity of the Taq polymerase become rate limiting. Moreover, this technology allows quantitation with a dynamic range of 7 logs and a sensitiv­ ity of less than 10 target sequences per sample. Three different types of chemistry are now applied to detect the accumulation of PCR products: fluorogenic probes, intercalating dyes, such as SYBR Green, and MGB (minor groove binding) probes. All three chemistries are based on the detection of changes in fluorescence levels which are proportional to accumulation of the PCR product. The first type of chemistry makes use of the 50 fluorogenic assay (Holland et al., 1991) and measures the amount of PCR product formed as a function of the cleavage of a fluorogenic probe containing a reporter dye and a quencher dye. These probes are called Taqman probes, or FRET (Föster Resonance Energy Trans­ fer) probes. While the probe is intact, the proximity of the quencher dye reduces the fluorescence of the reporter. During the extension phase of PCR, the probe anneals to its target sequence, if present, and is cleaved by the 50 nuclease activity of Taq DNA polymerase. The resulting separation of the reporter dye from the quencher generates increased fluorescence from the reporter. Since probe cleavage is depen­ dent on polymerization, the increase in fluorescence is proportional to the amount of PCR product formed. A variant of this chemistry, which has been applied more recently, makes use of modified internal probes that contain a reporter dye at the 50 end of the sequence, and at the 30 end a nonfluorescent quencher and a minor groove binder (MGB). There are various names for these probes including MGB probes and Blackhole quenchers. The minor groove binder increases the melting temperature (Tm) and allows for the use of shorter probes. The use of these probes is also dependent on the 50 nuclease activity of Taq polymerase to generate a signal from the reporter dye that is proportional to the accumulation of the PCR product in the 50 fluorogenic reaction. The advantage of this approach is the use of shorter probes and the absence of fluorescence from the quencher. The calculations on the increase of fluorescence derived from the reporter dye are more accurate since there is no contribution of fluorescence from the quencher, leading to an increased specificity. This also simplifies the detection of more than one target in a single tube (multiplex reactions), and up to four individual reactions can be monitored simultaneously with four different reporters. The second type of chemistry is based on the changes in fluorescence of a intercalating dye when it interacts with double-stranded DNA. Again, the accumulation of a PCR product is measured directly by the increase in fluorescence caused by the binding of the SYBR Green dye to double-stranded DNA. This type of reaction is independent of the sequence of the amplified product. It could thus detect amplification of non-specific or contaminant reactions. However, a dissocia­ tion or melting curve can be generated as a post-amplification step which determines the Tm of the PCR product and provides information on the amplifica­ tion and specificity. The advantage of this approach is the elimination of (costly) probes for each different target. The third type of chemistry is the use of molecular beacons (Tyagi and Kramer, 1996; Tyagi et al., 1998). These are also internal probes that contain the target 448

sequence in a hairpin loop between with reporter and quencher dyes on short flanking sequences. These probes will hybridize to the target, if present, which will disrupt the hairpin structure and allow for separation of the reporter and quencher dyes, resulting in a fluorescent signal. The fluorescent signal is in this application is recorded during the annealing phase of the PCR reaction. Alternative ways of applying and quantitating gene expression based on these three types of chemis­ tries have also been designed and include incorporation of fluochromes during amplification and addition of quenched probes as hairpin structures at the 50 or 30 end of primers (Didenko, 2001). In addition to the wide dynamic range and sensitivity of this type of quantitative PCR, a major advantage of the technology is that post-amplification steps are no longer required, making it very suitable for high throughput analyses. Indeed, 96 and 384 plate formats are currently in use. We will focus in this part on the application of Taqman and SYBR Green assays.

A. Methods The design of specific primer and probe pairs is dependent on the chemistry that is used in the application. In general, the melting temperature of the primers in all three chemistry reactions must be between 58°C and 60°C and the maximal difference between the Tm of forward and reverse primer is 2°C. The GC can be variable, but must be between 20 and 80%. More important is that there are no runs of four or more identical bases, in particular no G’s, and that the last five bases of the primers contain no more than 2 G/C residues. Primer length can be between 9 and 40 residues with an optimal length of 20. Complementary stretches at the 30 ends should be avoided to prevent primer–dimer formation, as well as hairpin structures in the primers, which decrease the efficiency of primer annealing. The Taqman probe should have a Tm of 68–70°C, which guarantees annealing of the probe prior to that of primers in the annealing phase of the PCR reaction. Again, runs of four or more identical bases should be avoided, as well as a G residue at the 50 end. Any G residue adjacent to the reporter dye will quench reporter fluores­ cence, even after cleavage. Preferably, the strand should be chosen where the number of C residues exceeds the number of G residues. MGB probes should have a Tm of 65–67°C, be as short as possible and contain less than 20 nucleotides. Since the PCR products do not have to be visualized on gel post amplification, the size of the amplicon can be short, in many cases just encompassing primer and probe sequences. This results in amplicon sizes that are generally less than 150 bp with a variable Tm of around 70°C. All these criteria are difficult to take into account without the use of computation. Primer Express (Perkin Elmer) and other software packages are available to design primer and probes that fit these criteria. Once primer and probe sequences are designed, an additional check for specifi­ city can be done by blast search against the non-redundant GenBank database (http://www.ncbi.nlm.nih.gov/). Primer design is an empirical process, as even after using the computational design algorithms, the primer and probe combina­ tion may not function efficiently. Thus, it may be worthwhile to design and test several primer/probe pairs for a particular target. 449

Measuring Human Cytokine Responses

1. Designing specific primers

Genes encoding cytokines or cytokine receptors, as well as many other genes in the genome, are usually composed of exons, containing coding sequences which are separated by non-coding intron sequences. To prevent amplification of genomic DNA, it is recommended to design one of the primers or the probe over an exon/ intron boundary. Primers and probes detecting human cytokine and cytokine receptor expression can be obtained as pre-developed assay reagents (PDAR’s) (Perkin Elmer). 2. Primer and probe purification

Due to the high constraints on the efficiency of the PCR reaction, it is worthwhile to purify primers and probe from contaminants. For primer purification a simple desalting step by spin chromatography is usually sufficient. Probes need to be purified by an additional HPLC (high-pressure liquid chromatography) step and preferably a PAGE (poly-acrylamide gel electrophoresis) step to eliminate any fluorochrome residues that are not attached and have to be checked for proper dual labelling. These procedures are usually performed by the manufacturer. Primers and probe concentrations are optimized for use in real-time PCR following design and purification. 3. Protocol

Primers from DNA synthesis facility (20–30 OD) are dissolved in H2O in 500 μl. Spin Sephadex G25 column (Microspin G-25 Columns: Amersham/Pharmacia 27-5325) for 1 min at 1700 × g. Change collecting tube, add 200 μl primer solution and spin Sephadex G25 column for 2 min at 1700 × g. Remove tube and read the OD of an aliquot which was diluted 100-fold. Calculate the molarity (M) according to the following formula: Absorbance ð260 nmÞ ðODÞ ¼ sum extinction coefficient contributions of each of the individual bases  cuvette pathlength  concentration dilution The extinction coefficients of the individual bases are A 15.200/C 7.050/G 12.010/T 8.400 The concentration in M is thus equal to 100 × OD / sum extinction coefficient con­ tributions of each of the individual bases when using a pathlength of 1 cm. Dilute primers to 45 μM. The concentration of primers and probe are important parameters for the efficiency of the PCR reaction. Apart from the design itself, these are the only parameters that are not standardized in a Taqman PCR reaction. Buffer components are optimized for use with a wide variety of primer/probe combinations with respect to Mg2+ concen­ tration in the Taqman and SYBR Green MastermixTM reaction buffers. Primer con­ centrations for Taqman applications are optimized in a checkerboard configuration at 450

50, 300 and 900 nM in duplo. Prepare in a tube 300 μl mastermix, 5 μl positive control target DNA (stock 10 μg ml–1), 12 μl probe (stock 10 μM, final 200 nM) and 83 μl H2O. In separate tubes make serial dilutions of forward and reverse primers from the 45 μM stock solution to yield final concentrations of 50, 300 and 900 nM. (Add 6 μl of stock primer to 44 μl H2O, from this add 16.3–32.6 μl H2O and from this add 8.3– 42.6 μl H2O.) Assemble 40 μl target/mastermix/probe and 10 μl of each dilution of forward and reverse primers in nine separate tubes, mix and transfer 25 μl to the reaction plate in duplicate wells. Run reactions and select the primer combination that results in the lowest CT value and the highest ΔRn (see below). Repeat this process with optimized primer concentrations, now varying the probe concentration from 100, 150, 200, 250 to 300 nM. Generally, primer concentrations of 900 nM and probe concentration of 250 nM give optimal results. 4. RNA preparation

There are two preferential ways to purify the RNA, depending mostly on the amount of available material.

Total RNA is isolated and purified using RNAzol [Quantum-Appligene] according to the manufacturer’s instructions and quantified by optical density readings. Pelleted cells are lysed with 1 ml of RNAzol and then 0.2 ml of chloroform is added. After centrifugation, the aqueous phase is recovered and precipitated with isopropanol (v/v). The pellet is resuspended in 10 µl of RNAse-free water. It is sometimes necessary to add glycogen or t-RNA as a carrier for better RNA recovery. 6. Less than 106 cells

Total RNA is prepared using an affinity column technique (Glassmax from Qiagen, Invitrogen). The Glassmax technique from Invitrogen is used according to the manufacturer’s instructions. Briefly, the cells are pelleted and homogenized into 400 µl of the lysis buffer, and then stored at –20°C until use. The homogenate is thawed on ice and pelleted 5 min at 20.000 x g with 280 µl of cold ethanol. The pellet is resuspended in 450 µl of the binding buffer and 40 µl of ammonium acetate and applied on a column, spun for 20 s at 20.000 x g and washed successively in the same manner with the wash buffer followed by 80% ethanol in water. The RNA is then eluted with 30–50 µl of RNAse-free water, previously heated at 70°C. 7. Reverse transcription

About 1 µg of total RNA (a lower concentration can be used, but for an easier comparison of the different samples, a constant number of cells is recommended) is resuspended in 20 µl of distilled water (H2O) and 1 µl of oligo-dT at 1 mg ml–1 is 451

Measuring Human Cytokine Responses

5. More than 106 cells

added together with 0.1 µl of 40 U ml–1 RNAsin (Promega). The samples are heated at 70°C for 10 min and cooled down at room temperature; the samples are briefly spun down in a microfuge after which 15 µl of the enzyme mixture (7 µl of 5X Superscript buffer (Invitrogen), 5 µl of 10 mM dNTP, 1.5 µl of 1 M DTT (Invitrogen), 0.1 µl of 40 U ml–1 RNAsin (Promega) and 1.5 µl of Superscript II (Invitrogen)) is added. The samples are then incubated at 42°C for 1 h and heat denatured at 95°C for 3 min, and after rapid cooling on ice, the volume is adjusted to 200 µl with H2O. The samples are then stored frozen at –20 to –80°C until use for RT-PCR amplification.

8. PCR amplification

Ten to fifty nanogram of cDNA per reaction is used as templates for quantitative PCR and analysis for the expression of human cytokine and cytokine receptor or transcription factor genes by the fluorogenic 50 -nuclease PCR assay using the GeneAmp 5700, ABI Prism 7700 or 7900 Sequence Detection Systems (PerkinElmer), Light Cycler (Roche), iCycler iQ (Biorad) or the Stratacycler (Stratagene). PCR reactions are assembled using 2× concentrated buffer solutions Universal PCR Master Mix (Taqman)- or SYBR Green PCR master mix (ABI)-containing enzymes, dNTPs and a inert fluorochrome (ROX) to yield final concentrations of 1× PCR buffer, 200 mM dATP, dCTP, dGTP and 400 mM dUTP, 5.5 mM MgCl2, 1.25 U AmpliTaq Gold DNA polymerase, Passive Reference I and 0.5 U Amp-Erase Ura­ cil-N-glycocylase (UNG). Forward and reverse primers are added at 900 nM each and FAM labelled probes at 250 nM in Taqman assays, and forward and reverse primers are used at 200 nM in SYBR Green assays. In a multiplex Taqman assay it is possible to measure additional targets in the same tube (actin, GAPDH, Ubiquitin, 18S rRNA), which can serve as internal controls for amount of input cDNA. For multiplex assays containing 18S rRNA detection reagents, forward primer, reverse primer and VIC labelled probe are all added at primer limiting concentrations of 50 nM in the PCR reaction. The thermal cycling conditions include at step 1 an incubation of 2 min at 50°C to eliminate any PCR-derived DNA contaminants by UNG, at step 2 an incubation of 10 min at 95°C to activate the ‘hotstart’ Taq Polymerase and at step 3, 40 cycles of amplification alternating between denaturing conditions at 95°C for 15 s and annealing-extension conditions at 55°C for 1 min.

9. Protocol

This protocol is for a multiplex taqman reaction with a final reaction volume of 25 μl. To ensure rigorous mixing and equal loading, 30 μl reactions are prepared, of which 25 μl is transferred to the final 96-well reaction plate. For 100 reactions assemble in a 5-ml polypropylene tube 1500 μl 2× buffer (Mastermix), 60 μl for­ ward primer target (45 μM Stock), 60 μl reverse primer target (45 μM Stock), 75 μl FAM labelled probe target (10 μM Stock), 15 μl forward primer control (10 μM Stock), 15 μl reverse primer control (10 μM Stock) and 15 μl VIC labelled probe 452

control (10 μM Stock) and 60 μl H2O. Mix well and distribute 18 μl per well with an electronic multipipettor (Biohit, Eppendorf or Gelman). Add 12 μl of sample DNA solution (containing 12–60 ng cDNA) or standard DNA solutions to the wells, mix well, spin plate 2 min at 770 × g and transfer 25 μl to the final reaction plate using a 12-channel electronic pipettor. Try to avoid as much as possible the formation of airbubbles during transfer. Close the plate with optical caps or adhesive optical cover and run. To make DNA standard solutions, start with a stocksolution of plasmid or other DNA containing the appropriate target sequence at 10 μg ml–1. In Eppendorf tubes make at least seven tenfold dilutions of the 10 μg ml–1 stock in H2O. From the 10 μg ml–1 dilution (third dilution) take 12 μl as the first point of the standard curve, which equals 100 pg in the final reaction. Do not use the first two dilutions as these are too concentrated for the detection system. You can make stocks of these plasmid dilutions. This protocol can be easily adapted for SYBR Green reactions by replacing the Taqman mastermix by SYBR Green Mastermix and addition of only 13.3 μl target forward primer and 13.3 μl target reverse primer with 274 μl H2O.

Following the amplification, the measurements of fluorescence intensity taken at each PCR cycle are collected and normalized by the software. The normalized reporter value Rn is obtained by dividing the emission intensity of the reporter dye (FAM, VIC, SYBR) by the emission intensity of the passive reference (ROX). This step allows for the correction of small changes in volume present in each individual well. If a positive reaction occurs, then the Rn will increase with an increase in cycle number. Rn+ is the Rn value of a reaction that contains all the components, whereas Rn– is the Rn value of an unreacted sample that is obtained from the early cycles in a PCR run (baseline) or from a reaction that did not contain template. The differ­ ence in Rn over time in the reaction is given by ΔRn = (Rn+) – (Rn–). ΔRn is thus the increase in fluorescent signal, specific for accumulation of the PCR product. Results of a quantitative PCR reaction are expressed as the Threshold cycle or CT values. The CT is the first cycle number at which the reporter fluorescence generated by cleavage of the probe passes a fixed threshold above baseline. The CT value is most accurately obtained from ΔRn values following log transformation. The software allows for easy visualization of Rn, ΔRn and log transformed ΔRn values which is needed to set the baseline and Threshold. The software will then give the CT values for the PCR reactions. It is important to realize that CT number is inversely correlated with the amount of target DNA in the sample; i.e. the CT equals 40 if no target is present. Using the CT values, it is possible to perform calculations on absolute and relative quantitation. When a standard curve of target DNA was run, the software calcu­ lates the absolute amount of target DNA present in the samples based on the CT values of standard curve and samples. These values can even be corrected based on the amount of internal control present by multiplication of normalized log trans­ formed CT values of the internal control reactions. Finally, fold expression over a 453

Measuring Human Cytokine Responses

10. Analyses of results and calculations

calibrator (e.g. a timepoint = 0, or an untreated sample) can be calculated without the need for absolute quantitation by the formula: fold = 21–ΔΔCT. The ΔΔ CT is equal to (sample target CT – sample internal control CT) – (calibrator sample CT – calibra­ tor internal control CT). A full derivation and theory of this latter can be found at http://www2.perkin-elmer.com/ab/about/pcr. These calculations can also be done using CT values obtained using SYBR Green if the internal control is run on a separate plate. One additional step that is recommended using SYBR Green assays is to analyse the dissociation curve of the PCR products and discount those that do not have the expected Tm.

tttttt IV. INTRACELLULAR CYTOKINE STAINING The method of staining with cytokine-specific mAbs for the analysis of intracel­ lular cytokines in suspension by immunofluorescence using an UV microscope has originally been described by Anderson et al. (1990) Subsequently, it was shown that the presence of intracellular cytokines could be detected using a FACS flow cytometer (Jung et al., 1993; Assenmacher et al., 1994) and recent improvements in the fixation procedure (Openshaw et al., 1995) and the use of Brefeldin as a protein transport inhibitor (Picker et al., 1995) have made the method even more suitable for multiparameter flow cytometry analysis. The advantage of this method over the measurement of cytokines in culture supernatants of activated cells is that it enables to determine the frequency of cytokine-producing cells, as well as kinetics of cytokine production. Moreover, in combination with cell surface staining, the method can be used to identify the phenotype of cytokine-producing cells in a population of non-separated cells, whereas the development of fluorochromes with different emission wavelength and flowcytometers, equipped with dual lasers, has permitted the simultaneous detection of several cytokines, provided the mAbs are directly conjugated with the appropriate fluor­ escent dyes. Critical parameters for successful intracellular cytokine staining include the choice of a proper fixation and permeabilization protocol, the inclusion of a protein transport inhibitor, cell type and activation protocol and kinetics of activation (Figure 1). The principle and sensitivity of the method depends on the availability of mAbs which recognize natural cytokines, in spite of a fixation and permeabiliza­ tion procedure. Fixation of the cells is required for subsequent treatment with detergent, and ideal fixatives preserve the morphology of the cells and antigeni­ city of the cytokines with minimal cell loss. Subsequent permeabilization of the cell membranes with the detergent saponin allows fluorochrome-labelled cytokine-specific mAb to penetrate through the cell membrane, cytosol and the membranes of the Golgi apparatus and endoplasmatic reticulum. It should be kept in mind, however, that the method provides a ‘snapshot’ of cytokine production and the observed frequency of cytokine-producing cells is affected by multiple variables, such as activation conditions and state of cell cycle of the cells. 454

Stimulate 106 cells/ml for 4 h

at 37°C, 5%CO2

Polyclonal activation of T cells with • crosslinked anti-CD3/anti-CD28 mAbs • anti-CD2 and anti-CD28 mAbs • TPA and A23187 or ionomycin Polyclonal activation of monocytes with • IFN-γ and LPS or TPA

Control cells are cultured in

medium for the same period of

time and treated similarly during

the protocol

Add Brefeldin A (10 mg/ml)

and incubate for

an additional 2 h

Agents which block intracellular protein transport have doseand time-dependent cytotoxic effects. It is not recommended to include Brefeldin in the cultures >6 h. Monensin will induce both intracellular TNF and IL-1 production within 30 min of stimulation

Harvest the cells, wash with

PBS/2% FCS and stain for

surface molecule expression

Activation with TPA causes a downregulation of CD4 expression. Some mAbs to cell surface Ag may not bind to fixed, denatured proteins, and cell surface staining should be performed prior to fixation. Protect cells from light during staining procedures. Non-specific FcR-binding can be inhibited by preincubation of the cells with 2% Ig from species in which mAb was generated Cells should be thoroughly washed to remove any protein, prior to fixation

Fix the cells for 20 min

with 2% formaldehyde

Wash twice with PBS/2% FCS

Vortex gently to avoid aggregation of the cells. Fixed cells can stored at 4°C for at least a week and for several weeks following storage at –80°C. When methods involving simultaneous fixation/ permeabilization are used, stored cells should be re­ permeabilized prior to staining

Permeabilize the cells with

saponin (0.5% in PBS/2% FCS)

for 10 min in the presence or

absence of relevant animal

serum

Because its effects are reversible, saponin should be maintained in the mAb solution and staining buffer to prevent closure of the membranes. Cells, spun in the presence of saponin, will not show clear. pellet. However, if properly spun, no cells loss will occur

Stain with cytokine­ specific mAb diluted in PBS/saponin/FCS

Non-stimulated, identically treated, cells serve as negative staining control. Each mAb should be titrated for optimal staining on stimulated cells and absence of staining on control cells. Specificity of staining can be controlled by • Preicubation of the anti-cytokine mAb with recombinant cytokine • Preincubation of the cells with unlabelled mAb, followed by staining with the same, fluorochrome-labeled, mAb

Wash with PBS/2%FCS

Saponin should be removed prior to flow cytometric analysis

Analyse on the FACS

If two- or more colour analysis is performed, PMT voltage and

compensation should be set on cell surface controls.

Always include a positive control (T cell clone) which should be

prepared, aliquotted and stored at –80°C.

Quadrant markers should be set on the negative control (non-

stimulated cells)

Perform control analysis using UV microscope

Figure 1. Flowchart for the intracellular staining procedure

455

Measuring Human Cytokine Responses

Wash the cells with PBS

A. Reagents and Equipment • Washing buffer. PBS without Ca2+ and Mg2+, supplemented with 2% heatinactivated FCS and 0.1% (w/v) sodium azide (NaN3) (see note 1). Adjust buffer to pH 7.4 and store at 4°C. • Permeabilization buffer. Washing buffer, supplemented with 0.5% saponin (Sigma; S7900). Make stock solution of 10% saponin in PBS (pH 7.4), dissolve at 37°C, filter solution through a 0.2 μm filter and store at 4°C (see note 1). • Formalin (=37% formaldehyde solution, Sigma F1635). Dissolve 10.8 ml of formalin in 89.2 ml PBS, filter to remove particles and store at 4°C (see note 1). • Cytokine-specific mAbs (preferentially conjugated directly with a fluorochrome (fluorescein isothiocyanate (FITC) phycoerythrin (PE), or Cy5)). A list of mAbs suitable for intracellular staining is shown in Table 3. • Second-step fluorochrome-labelled Abs. Horse–anti-mouse IgG-FITC/PE (Vector; FI 2000/EL 2000). Rabbit–anti-rat IgG-FITC (Vector; FI 4000). • For analysis by FACScan flowcytometer. Vortex; centrifuge with rotor to spin microtitre plates; 96-well V-bottom plates (Falcon, BD Biosciences); FACS tubes (BD Biosciences).

B. Protocol: Staining for Flow Cytometry The procedure for activating cells is given on page 429 (see notes 2 and 3). editor: page of activation procedure) If cell surface staining is performed, start with step 1; for intracellular cytokine staining without cell surface staining, start with step 7 (see note 4). 1. Transfer the stimulated cells to 15-ml centrifuge tube and wash twice with 1 ml of ice-cold washing buffer. Washing with larger volumes will result in cell loss. Spin the cells at 200 × g for 5 min. 2. To block non-specific binding of FcR, add 20 μl of 2% serum (from same animal species as the mAbs used for the immunofluorescence staining) to the cell pellet and incubate cells for 15 min. 3. Wash cells twice with 1 ml of cold washing buffer. 4. Add 20 μl of directly-conjugated anti-cell surface mAb to the cell pellet. 5. Incubate for 15 min on ice. 6. Wash cells twice with 1 ml ice-cold PBS and proceed to step 8. 7. Transfer the stimulated cells to 15-ml centrifuge tube and wash twice with icecold PBS. 8. Resuspend the cells at 2 × 106 cells ml–1 in cold PBS and add an equal volume of 4% formaldehyde (see note 5). Mix well to prevent aggregates. 9. Incubate for at least 20 min at room temperature. 10. Spin the cells at 200 × g for 5 min and wash once more with 1 ml of cold PBS. At this point the cells can be resuspended in PBS and stored at 4°C in the dark for 1 week or aliquoted and stored at –80°C for at least one month prior to analysis (see note 6). 456

Table 3. Human cytokine-specific mAbs for intracellular staining Detection mAb

Cytokine

mAb

Isotype

IL-1β IL-2 IL-2 IL-3 IL-3 IL-4 IL-4 IL-4 IL-5 IL-6 IL-6 IL-8 IL-10 IL-10 IL-10 IL-12p40 IL-12p70 IL-13 IL-13 IL-13 IL-15 G-CSF GM-CSF TNF-a TNF-a TNF-a IFN-g IFN-g IFN-g IFN-g IL-17 IL-22

B-A15 MQ1-17H12 BG-5 BVD8-3G11 BVD3-If9 8D4 MP4-25D2 3010.2 JES-39D10 MQ2-6A3 B-E8 B-K8 JES8-12G8 B-N10 JES3-19F1 B-P24 B-T21 JES10-30F11 JES8-5A2 B-B13 B-T15 BVD13-3A5 BVD2-21C11 BVD11-37G1 B-D9 MP9-20A4 B27 4S.B3 25723.11 B-B1 41802 142928

mIgG1 rIgG2a mIgG1 mIgG2a mIgG2a mIgG1 rIgG2a mIgG1 rIgG2a rIgG2a mIgG1 mIgG1 rIgG2a mIgG1 mIgG1 mIgG1 mIgG1 rIgG2a rIgG2a mIgG1 mIgG1 rIgG1 rIgG2a mIgG1 mIgG1 rIgG1 mIgG1 mIgG1 mIgG1 mIgG1 mIgG1 mIgG1

Sourcea

1 2 1 2/3 2/3 3 2/3 3 2/3 2/3 1 1 2/3 1 2/3 1 1 2 2/3 1 1 2/3 2/3 2/3 1 3 1 3 3 1 4 4

m: mouse mAb, r: rat mAb.

a Abs sources are as follows: (1) GenProbe, Besançon, France. (2) Generated at DNAX Research Institute, Palo Alto,

CA, USA; relevant hybridomas can be obtained via American Tissue Culture Collection; permission must be

obtained from DNAX for acquisition of cell lines from this source. (3) BD Biosciences/PharMingen. (4) R&D Systems.

11. Resuspend the cells in washing buffer and transfer ± 105 cells per well of a 96­ well V-bottom microtitre plate and spin at 200 × g for 2 min. 12. Remove supernatant by flicking the plate, blot the plate dry on paper tissue and resuspend the cells by gently vortexing. 13. Add 150 μl of permeabilization buffer to the cells and incubate for 10 min at room temperature. A final concentration of 2% serum (as described under step 2) can be added to block non-specific FcR binding of mAbs. 14. Spin the cells at 200 × g for 2 min and remove supernatant. 457

Measuring Human Cytokine Responses

Coating mAb

15. Add 20 μl of cytokine-specific mAb solution to the cell pellet (see note 7). 16. Incubate cells for 20 min at room temperature. 17. When using non-conjugated mAbs, proceed to step 18; for FITC, PE or Cy5-conjugated-conjugated mAbs, proceed to step 22. 18. Add 150 μl of permeabilization buffer, centrifuge the plate at 200 × g for 2 min, remove supernatant and resuspend the cells. 19. Repeat washing step. 20. Add 20 μl per well of an appropriate dilution of the second conjugated mAb. 21. Incubate cells for 30 min at room temperature. 22. Add 150 μl of permeabilization buffer, centrifuge the plate at 200 × g for 2 min, remove supernatant and resuspend the cells. 23. Repeat washing step. 24. Wash once with washing buffer, resuspend in 200–300 μl PBS and analyse on the FACS flowcytometer as soon as possible. 25. For instrument control and compensation setting, refer to the manufacturer’s instructions (see Chapter 1, “Phenotyping and separation of leucocyte popula­ tions based on affinity labelling” of Volume 32 and notes 6–9).

C. Notes and Recommendations 1. NaN3 is known to be toxic, saponin is an irritant and formaldehyde is a suspected carcinogen. Avoid contact with skin, eyes and mucous membranes. Dispose of formaldehyde in container for treatment; do not put formaldehydecontaining waste in sink. Always wear gloves. 2. Since cytokines are produced with different kinetics, optimal time points for the analysis of activated cytokine-producing cells may vary, depending not only on the type of cytokine to be analysed, but also on the mode of stimulation. The peak production of most cytokines, including IL-2, IL-4 and TNF-a, by T cell clones, following stimulation with TPA and A23187, is within the first 6–7 h, whereas others, such as IFN-g and IL-10, have more long-lasting kinetics. IL-13 is produced early (2 h) following activation, but this cytokine also continues to be produced up to 72 h. Analysis of the cells 4–6 h after activation results in a characteristic staining of the Golgi apparatus/endoplasmatic reticulum, which is bright for cytokines such as IL-2, TNF-a and IFN-g. 3. The addition of agents which inhibit cellular transport systems, such as Brefel­ din A or Monensin, results in an accumulation of newly synthesized protein in the Golgi complex. Although the cellular integrity changes following treatment with intracellular protein transport inhibitors, resulting in a disappearance of Golgi staining and a homogeneously fluorescing cells, their use generally gives a brighter staining pattern and enables the detection of intracellular cytokines that stain only weakly with anti-cytokine mAbs, such as IL-4 and IL-5. However, since these compounds have dose- and time-dependent cytotoxic effects, their use should be limited. Brefeldin A is less toxic than Monensin and should be added to the cultures between 2 and 6 h before harvesting the cells. 4. The staining procedure for flowcytometric analysis is performed in 96-well V-bottom microtitre plates. However, since it is preferable to fix the cells in 458

459

Measuring Human Cytokine Responses

15-ml centrifuge tubes, it is recommended to perform staining of cell surface molecules in tubes, followed by fixation and transfer of the cells to microtitre plates. All steps during cell surface staining are carried out at 4°C, in the absence of saponin. Since some mAbs may not bind to fixed, denatured, protein cellsurface staining should be carried out before fixation of the cells. Staining for intracellular cytokines is performed at room temperature in the presence of saponin. All incubation periods during the staining procedures are carried out in the dark. • Make the Ab dilutions in PBS/2%FCS/NaN3/0.5% saponin. For each mAb, the optimal titre has to be determined. Generally, final concentrations of mAbs are 1–5 μg per 106 cells. PE-conjugated mAbs are not recommended for analysis on a UV microscope, because of the quick fading of the dye: use Rhodamin-conjugated Abs instead. • Centrifugation of the Abs at high speed in a microfuge to remove aggregates will improve the quality of the staining. • If non-conjugated anti-cytokine mAbs are used, obtained from different species, two-colour analysis can be performed. Although cytokine-specific staining signals generally tend to be higher, often increased fluorescence backgrounds are observed, requiring rigid blocking of non-specific mAb binding. 5. Optimal results have been obtained with formaldehyde (Openshaw et al., 1995), and although this fixative is less efficient to cross-link and change the tertiary structure of protein as compared to glutaraldehyde, its effect are more gentle and fixation with formaldehyde generally preserves a high degree of antigenicity. 6. Positive control. Samples of a resting and activated T clone with a Th0 cytokine production profile can be fixed, aliquotted in PBS, stored at –80°C for several weeks and used in the staining procedure as negative control (to adjust flowcytometer instrument settings) and positive controls, respectively. 7. Negative control. Non-stimulated cells, which have been identically treated dur­ ing the staining procedure, serve as negative control. Prior to its use in the staining procedure, each anti-cytokine mAb should be titrated at a concentra­ tion which gives optimal staining on activated cells and absence of staining on non-stimulated control cells. Furthermore, it is recommended to use one of the following controls to confirm specificity of the staining: • Ligand-blocking control. Pre-incubate mAb with excess of recombinant cyto­ kine in permabilization buffer at 4°C for 30 min. It is recommended to use at least a 50-fold Molar excess of cytokine over mAb. Proceed to step 15 of staining protocol. • Non-conjugated mAb control. Pre-incubate cells after step 14 of staining proto­ col with non-conjugated mAb, diluted in permabilization buffer, at 4°C for 30 min, wash cells twice and proceed to step 15 of staining protocol. • Isotype control. Use an irrelevant mAb of the same isotype as the anti-cytokine mAbs in the staining procedure. Isotype controls can be used to adjust instrument settings, including quadrant markers and compensation of flowcytometer. In cases where isotype controls give brighter staining than anti­ cytokine mAb, rely on non-activated cells as negative control.

8. It should be stressed that when the intracellular staining method, using flow cytometry, is introduced in the laboratory, the results should be confirmed by immunofluorescence using an UV microscope. Intracellular cytokine staining of cells that have been activated less than 6 h in the absence of Brefeldin A will result in distinct staining of the Golgi apparatus and endoplasmatic reticulum, whereas after longer activation periods also cell surface cytokine staining may be detected. Microscopic analysis of cells, activated in the presence of Brefeldin A, shows homogenous staining throughout the cytoplasm, as a result of disin­ tegration of Golgi apparatus and endoplasmatic reticulum.

tttttt V. ELISA In this section, the antibody sandwich ELISA system is described which is used for the detection of cytokines present in the culture supernatants of in vitro activated cells of the immune system or in clinical samples, such as serum, plasma or bronchoalveolar fluid. Although generally not as sensitive as a bioassay, the major advantage of the ELISA is its specificity, enabling the detection of soluble proteins in complex mixtures, which often have overlapping or, by contrast, counter-regulatory activities and which are therefore extremely difficult to detect, based on their biological activities alone. To measure a cytokine in solution, plates are coated with an anti-cytokine mAb which functions as a catcher to bind the cytokine. After removal of unbound cytokine, a second anti-cytokine mAb is added (detection mAb), which is usually biotinylated and which recognizes a different epitope on the cytokine, followed by a washing step and the addition of an enzyme–streptavidin conjugate. The use of such a biotin–streptavidin conjugate in the assay enables amplification of the signal. After removal of unbound conjugate, substrate is added, the hydrolysis oxidation of which by the enzyme–con­ jugate is proportional to the amount of cytokine present in the solution.

A. Reagents and Equipment • Cytokine-containing supernatants, cytokine-specific capture mAb; biotinlabelled, cytokine-specific tracer mAbs; PBS; dH20. • Culture medium to dilute cytokine-containing samples and cytokine standards: Coating buffer: Carbonate–bicarbonate buffer pH 7.2 to 7.4. Washing buffer: PBS, supplemented with Tween 20 (0.05%). Tween buffer: PBS, supplemented with bovine serum albumin (BSA) (0.1%) and Tween 20 (0.02%). Blocking buffer: PBS, supplemented with BSA (2%). Conjugate: Strepavidine–alkaline phosphatase (AP) conjugate (Southern Biotechnology Associates: 7100-04) or streptavidin–HRP (Prozyme: CJ30H). ELISA substrate: Sigma 104 phosphatase substrate (Sigma: 104-0) or ready-to-use 3,30 ,5,50 -tretramethylbenzidine (TMB) (Moss: TMBUS). Substrate buffer: Diethanol amine: 97 ml in 800 ml dH20. 460

0.2 g NaN3. 0.1 g MgCl2.6H2O.

Adjust to pH 9.8 with HCl and adjust volume to 1 l with H2O.

• H2O2; PVC U bottom 96-well plates; Immunolon I U bottom 96-well plates; Immunolon II U bottom 96-well plates (Dynatech: 011-010-3450); Microtitre plate reader-spectrophotometer with 405 nm filter or spectro fluorometer (Dynatech: 011-970-1900) with 365 nm excitation filter and 450 nm emission filter. All reagents should be at room temperature before use in the ELISA. The optimal working concentrations of all Abs should be determined for each ELISA. Usually catcher Ab concentrations range from 1 to 10 µg ml–1. A list of mAbs and polyclonal Abs available for the detection of cytokines by ELISA is shown in Table 4. Table 4. Human cytokine-specific mAbs for ELISA Detection mAb

Cytokine

Designation

Designation

IL-1a IL-1β IL-2 IL-2 IL-2 soluble (s) CD25 IL-3 IL-4 IL-4 sIL-4R sIL-4R IL-5 IL-5 IL-6 IL-6 sIL-6R SIL-6R IL-7 IL-7 IL-8 IL-8 IL-10 IL-10 IL-12p40 IL-12p40 IL-12p40 IL-12p70 IL-13 IL-13 IL-15

B–Z3 B–A15 MQ1-17H12 Goat polyclonal Ab 5344.111 BG-3 BVD8-3G11 B-R14 8D4-8 S456C9 Hil4R-M10.1 JES-39D10 Goat polyclonal Ab MQ2-39C3 B-E8 B-N12 M5 BVD10-40F6 B-N18 B-K8 G265-5 JES3-19F1 B-N10 B-P40 C8.3 20C2 B-T21 JES10-5A2 B-B13 Goat Polyclonal Ab

B-Z5 B-Z8 BG-5 BG-5 B333-2 B-F2 BVD3-IF9 B-G28 MP4-25D2 BB4N1 HIL4R-M8.2.2 JES1-5A10 B-Z25 MQ2-13A5 B-E4 B-R6 M182 BVD10-11C10 B-S16 Rabbit polyclonal Ab G265-8 JES3-12G8 B-T10 B-P24 C8.6 (p40/p70) C8.6 (p40/p70) B-P24 B69-2 B-P6 B-E29

Sourcea coating/detection 1/1 1/1 4/1 1/1 2/2 1/1 4/4 1/1 2/2 1/1 2/2 4/4 1/1 4/4 1/1 1/1 2/2 4/4 1/1 1/1 2/2 2/2 1/1 1/1 2/2 2/2 1/1 4/4 1/1 1/1 (Continued)

461

Measuring Human Cytokine Responses

Coating mAb

Table 4.

(Continued )

Coating mAb

Detection mAb

Cytokine

Designation

Designation

IL-17 IL-17A IL-17A/F IL-17F IL-22 IL-23 IL-27 IL-31 IL-33 IL-18 IL-21 G-CSF GM-CSF TNF-a TNF-a LT-a/TNF-β LT-a/TNF-β IFN-g IFN-g OSM

B-C49 41809 B-C49 B-G46 142906 B-Z23 B-G44 B-S31 B-L33 Goat polyclonal Ab J148-1134 BVD13-3A5 BVD2-23B6 MP9-20A4 28401 359-238-8 5807 A35 B-B1 17001

B-B51 Goat Ab B-F60 B-F60 142928 B-F43 B-G49 B-A51 B-S33 Goat polyclonal Ab I76-539 BVD11-37G10 BVD11-21C11 GMO1–1782 Goat Ab 359-81-11 Goat Ab B27 B-G1 Goat Ab

Sourcea coating/detection 1/1 3/3 1/1 1/1 3/3 1/1 1/1 1/1 1/1 1/1 2/2 4/4 4/4 4/4 3/3 2/2 3/3 1/1 1/1 3

a

This list is a selection of (m)Ab for ELISA purposes, but is not exhaustive. (1) Diaclone, Besançon, France; (2) BD Biosciences; (3) R&D Systems; (4) generated at DNAX Research Instititute, Palo Alto, CA, USA; relevant hybrido­ mas can be obtained via American Tissue Culture Collection; permission must be obtained from DNAX for acquisition of cell lines from this source.

B. Protocol The procedure for activating cells is given on page 429. 1. Coat 96-well assay plate with 100 μl catcher Ab, diluted in carbonate buffer. 2. Seal plate with lid or parafilm and incubate for 2 h at 37°C or overnight at 4°C. Plates can be stored in a sealed pouch with a dessicant bag at 4°C. Before storage, a blocking solution should be added. Avoid storing plates for periods longer than several weeks. 3. Wash wells three times with washing buffer and flick plates dry on absorbent tissue. 4. Add 300 μl of bocking buffer, incubate for 30 min and flick plates dry on absorbent tissue. Do not wash. 5. Dilute the samples and cytokine standard (aliquots stored at –80°C) in Tween buffer, mix well and add 100 μl per well in duplicate at appropriate dilutions. • Standard curve: dilute in separate plate in 1:2 dilutions to cover a 1000–20 pg ml–1 decreasing range and add 100 μl per well in duplicate. • Unknown samples: dilute as needed (see notes) and add 100 μl per well.

462

6. Incubate plates for overnight at room temperature. When incubation time for the standard or sample is unknown, it is recommended to incubate the plate overnight at 4°C. Alternatively, the biotinylated tracer Ab together with the standard should be incubated overnight at 4°C. 7. Wash three times with washing buffer and flick plates dry on absorbent tissue. 8. Dilute the biotin-conjugated anti-cytokine detection mAb in Tween buffer and add 100 μl per well. 9. Incubate for 1–2 h at room temperature. 10. Wash three times with PBS and flick plates dry on absorbent tissue. 11. Prepare strepavidin–alkaline phosphatase or streptavidin–HRP conjugates in Tween buffer and add 100 μl per well. 12. Incubate for 1 h at room temperature. 13. Wash three times with washing buffer and flick plates dry on absorbent tissue. 14. Prepare the ELISA substrate (Dilute 1 mg ml–1 ELISA substrate in 100 ml of substrate buffer. Prewarm at 37°C and add 100 μl per well. TMB substrate is ready to use and should be at room temperature before addition.) 15. Incubate for 30 min at 37°C to let colour develop and read OD at 405–430 nm using a spectrophotometer or spectrofluorometer (Dynatech). For TMB, block the reaction with 1N sulphuric acid and read absorbence at 450 nm with a reference filter set at 630 nm. 16. Use the Softmax program (Molecular Devices Corporation) or comparable program to analyse the data.

It is recommended to make several dilutions of cytokine-containing supernatants to be able to measure at the linear part of the standard curve. It is important to note that the presence or absence of detectable levels of cytokines in clinical samples does not always correlate with their functional activ­ ity. Cytokines are often bound to non-specific serum proteins or to soluble cytokine receptors, which will sequester free cytokine from the peripheral circulation and might result in a decrease in detectable cytokine levels, as measured with ELISA. The biological significance of free versus complexed cytokines however is not yet clear. In addition, circulating anti-cytokine Abs, notably in the serum of those who have received cytokine or anti-cytokine therapy, may decrease levels of free cyto­ kine, although they may still be detectable by cytokine-specific mAbs. Conversely, cytokine receptor antagonists may specifically compete with the cytokine for binding to its receptor. For example, despite high levels of IL-1 in certain serum samples, possible receptor occupancy by IL-1RA will prevent IL-1-mediated responses and therefore will prevent a meaningful analysis about its functional activity (Ahrend, 1993). This situation is similar for IL-18 and the IL-18BP (Novick et al., 1999). The half-life time of many cytokines in serum is very short, generally in the range of minutes, which is one of the reasons that cytokine levels in the peripheral circulation are usually very low in healthy individuals, as measured by ELISA. However, certain cytokines, notably IL-1, IL-6, IL-10 and TNF-a, may be detectable 463

Measuring Human Cytokine Responses

C. Notes and Recommendations

in the serum following sepsis, trauma or an acute or chronic inflammatory state. It should however be kept in mind that, due to existing cytokine networks, even in above-mentioned pathological situations, the presence in excess of certain cyto­ kines may result in the suppressed production of another and a failure to detect the latter cytokine. Due to the possible presence of proteases or other (unknown) factors, in clinical samples, as well as culture supernatants, cytokines may be unstable and subject to degradation. Therefore, it is recommended to aliquot and store samples for cyto­ kine measurement at –80°C prior to analysis, especially when prolonged periods of storage are required. Serum may contain certain factors such as heterophilic Abs that have the ability to cross-bridge the capture Ab attached to the solid phase with the biotinylated tracer Ab which may result in false-positive results. The reported incidence of human anti-mouse Abs in the serum of normal individuals is around 3%. Human anti-mouse Abs develop with high incidence in subjects who are in contact with animals or animal products or in patients who have been treated with murine mAbs. Rheumatoid factors (RFs) are polyreactive IgM Abs produced by a subset of B lymphocytes and bind to the Fc portion of the IgG molecule. RF may also interfere in the ELISA assay by bridging the tracer to the capture Ab in the absence of analyte. Another important issue in immunoassays in general is the calibration of the assay. As the same amount of cytokine will not give the same signal in different assays, it is very important to calibrate the standard to the available international standard in order to be able to compare results. One last point that should be emphasized is that different monoclonal or polyclonal Ab pairs sometimes give different results which are likely to depend on the different epitopes that are recognized by these Abs. For example, some epitopes might be hidden or denatured by certain cytokine­ binding proteins or proteases. Furthermore, the variable level of glycosylation of some cytokines, such as IL-6, may also interfere with the capacity of Abs to fully recognize the natural cytokine. Finally, the capacity of various Abs to detect monomers rather than naturally occurring polymers (TNF-a, IFN-g, IL-10, IL-12) should also be taken into account when considering the varia­ bility between immunoassays.

tttttt VI. ELISPOT ASSAY The ELISPOT assay is based on immuno-enzyme technology originally developed for the enumeration of Ab-secreting cells (Czerkinsky et al., 1983; Sedwick and Holt, 1983; Czerkinsky et al., 1988). The assay was subsequently adapted to mea­ sure cytokine production at the single cell level. The ELISPOT assay is easy to perform and requires, compared to other approaches such as limiting dilution or intra-cellular staining, a minimum in vitro cell manipulation, allowing analysis of cells close as the in vivo situation. It can be used successfully to measure ongoing cytokine production by freshly isolated mononuclear cells. The technique is designed to determine the frequency of cytokine-producing cells under a given 464

A. Reagents and Reagent Preparation • Capture anti-cytokine mAbs. • Biotin-conjugated anti-cytokine detection mAbs.

465

Measuring Human Cytokine Responses

stimulation, and it enables the follow-up of these frequencies during a treatment and/or different stages of disease. Cells can either be stimulated directly in wells containing Ab-coated mem­ brane (direct method) or first stimulated for 6 h in 24-well plates, harvested and transferred to ELISPOT plates (indirect method). The choice between the two methods depends on the type of cell to be assayed, the expected frequency of cytokine-producing cells and the mode of stimulation. Stimulation of cells with immobilized anti-CD3 mAb can only be carried out using the indirect stimulation procedure. The technique includes the following steps: Single cell suspensions are dis­ tributed in membrane-bottomed wells previously coated with an anti-cytokine Ab. During cell stimulation, locally produced cytokine molecules will be caught by surrounding capture Ab and accumulate in the close environment of the cell. After cell lysis, trapped cytokine molecules are revealed by a secondary biotinylated detection Ab, which is in turn recognized by streptavi­ din conjugated to alkaline phosphatase. The enzymatic reaction in the presence of the substrate BCIP/NBT produces a precipitate. Cytokine secreted by indi­ vidual cells is visualized by sharp blue/purple spots that can be counted on an inverted microscope or a reading system, allowing the numeration of cytokine producing cells under a given stimuli. The recently developed ELISPOT assays allow to monitor the simultaneous production of two or more cytokines by the same cell population (Figure 2). In this method, Polyvinylidine Fluoride (PVDF) filters in 96-well plates are coated with two different Abs each one specific for one of two cytokines to be analysed. After blocking of non-specific binding sites on the membrane, cells are incubated for the appropriate length of time, in the presence of a stimuli i.e. protein, peptide or tumour cells. After lysing of the cells, cytokine molecules trapped by the catcher Ab are recognized by two differently tagged secondary Abs. For one cytokine, Ab are labelled with biotin; for the other, Abs are conjugated with FITC molecules. These Abs are in turn detected by streptavidin–alkaline phosphatase conjugates, or FITC peroxydase conjugates for the biotinylated and by FITC conjugated Ab, respectively. Finally, addition of BCIP/NBT and AEC, the respective substrates of alkaline phosphatase and peroxidase, will result in blue/purple and red/brownish spots in the presence of their enzyme, allowing the visualization of cytokine molecules produced by the cells. In a recent development, the enzymatic revelation system has been replaced by fluorescent dyes conjugates such as streptavidin–PE and anti-FITC green fluorescent dye. Fluorescent spots are then visualized under a UV light beam on a UV detection system.

1

Polyclonal activation of T cells with • crosslinked anti-CD3/anti-CD28 mAbs • anti-CD2 and anti-CD28 mAbs • TPA and A23187 or ionomycin Polyclonal activation of monocytes with • IFN- and LPS or TPA

Cell

Immobilized a-CD3 and soluble a-CD28 mAbs 1

2

Activated cell

4

Activated cell

Stimulate 106 cells/ml for 6 h at 37°C, 5%CO2, or use fresly isolated clinical material Catcher mAbs specific for cytokine A or cytokine B

2 Transfer cells to antibody coated PVDF-filter containing-wells and incubate for additional 18 h

3

Activated cell

3 Tracer mAb specific for cytokine A

Wash plates to remove cells and incubate with mAb, reactive with a different epitope on cytokine A and coupled to enzyme of fluorochrome

Enzyme 1

Wash plates and incubate with mAb, reactive with a different epitope on cytokine B and coupled to enzyme of

fluorochrome different from

those on mAb anti-cytokine A

Tracer mAb specific for cytokine B

4

Enzyme 1

Enzyme 2

3 and 4 can be carried ) ( Stepsout simultanously Colourless substrate 1

Coloured substrate 1

5 Enzyme 1

5

Wash plates and add

specific substrate for

enzyme A

6

Wash plates and add specific substrate for

enzyme B

Colourless substrate 1

Coloured substrate 1

6 Enzyme 1

Analyse cytokine-producing cells,

by quantifying numbers of single or

dual colour spots by optical densitity

or by detection of specific

fluorescence

Figure 2. Flowchart for the ELISPOT procedure

466

Enzyme 2

Colourless substrate 2

Coloured substrate 2

Enzyme 2

• FITC and PE-conjugated anti-cytokine detection mAbs (for dual cytokine ELISPOT). • Streptavidin alkaline phosphatase conjugate (Prozyme). Dilute 1/1000 in PBS–1% BSA. • Anti-FITC HRP-conjugated pAb (CTL, Diaclone, MABTECH; for dual ELI­ SPOT). Dilute 1/500 in PBS–1% BSA. • AEC substrate buffer (for dual ELISPOT). For one plate, mix 1 ml of AEC buffer A with 9 ml of H2O. Then add 200 µl of AEC buffer B. • BCIP/NBT substrate buffer. • PBS, BSA (Sigma: A2153), ethanol (35% in H2O), Tween 20 (0.05% in PBS). • 96-well PVDF filter-containing ELISPOT plates (CTL, Millipore MultiScreen; MSIPN4510) are recommended. • ELISPOT reading system (AELVIS, Zeiss or CTL).

B. Reagent Storage • If not used within a short period of time, reconstituted detection Ab should be aliquoted and stored at –20°C. Under these conditions, the reagent is stable for at least 1 year. • Substrate buffers to be stored at 4°C. • Streptavidin–alkaline phosphatase as well as anti-FITC HRP conjugates should be stored at 4°C.

The procedure for activating cells is given on page 429 (see notes 2 and 3). 1. Incubate PVDF filter-containing wells of an ELISPOT plate with 100 µl of 35% ethanol for 30 s at room temperature. 2. Empty wells (flick the plate and gently tap it on absorbent paper). Wash three times with 100 µl PBS. 3. Pipette 100 µl of each of the capture mAbs in 10 ml of PBS. Mix and dispense 100 µl into each well, cover the plate and incubate overnight at 4°C. 4. Empty wells and wash plate once with 100 µl of PBS. 5. Dispense 100 µl of complete culture media into wells, cover and incubate for 2 h at room temperature. 6. Empty wells and wash plate once with 100 µl of PBS. 7. Add to a well of an ELISPOT plate between 1.105 and 2.5 × 104 of pre-activated cells (indirect method) or cells to be activated with the appropriate stimulator (direct method) in a final volume of 100 µl. Cover the plate with a plastic lid and incubate for 10–15 h at 37°C, 5% CO2. During incubation do not disturb the plate. 8. Empty wells, add 100 µl of PBS–0.05% Tween 20 and incubate for 10 min at 4°C. 9. Empty wells and wash plate three times with 100 µl of PBS–0.05% Tween 20. 467

Measuring Human Cytokine Responses

C. Protocol

10. For one plate, dilute 100 µl of cytokine 1 detection Ab and 100 µl of cytokine 2 detection Ab into 10 ml of PBS containing 1% BSA. Dispense 100 µl into wells, cover the plate and incubate 1 h 30 min at room temperature. 11. Empty wells and wash three times with 100 µl of PBS–0.05% Tween 20. 12. For one plate dilute 20 µl of anti-FITC HRP and 10 µl of streptavidin– alkaline phosphatase conjugates into 10 ml of PBS–1% BSA. Dispense 100 µl of the dilution into wells. Seal the plate and incubate for 1 h at room temperature. 13. Empty wells and wash three times with 100 µl of PBS–0.05% Tween 20. 14. Peel off the plate bottom and wash three times both sides of the membrane under running distilled water. Remove all residual buffer by repeated tapping on absorbent paper. 15. Prepare AEC buffer (see reagents preparation) and add 100 µl of solution to each well. 16. Let colour reaction proceed for 5–15 min at room temperature. When the spots have developed, remove the buffer and dispose of appropriately. 17. Wash both sides of the membrane thoroughly with distilled water and care­ fully remove residual buffer by tapping the plate on absorbent paper. 18. Dispense 100 µl of ready-to-use BCIP/NBT buffer into wells. 19. Let the colour reaction proceed for about 5–15 min at room temperature. When spots have developed, empty the buffer into an appropriate tray. 20. Wash thoroughly both sides of the membrane with distilled water 21. Dry the membrane by repeatedly tapping the plate on absorbent paper. Store the plate upside down to avoid remaining liquid to flow back onto the mem­ brane. Read spots once the membrane has dried. Note that spots may become more distinct following incubation of the filters at 4°C for 24 h. Store the plate at +4°C away from direct light.

D. Notes and Recommendations 1. AEC and BCIP/NBT buffers are potentially carcinogenic and should be disposed off appropriately. Caution should be taken while handling those reagents. Always wear gloves. 2. While using MSIPN4510 plates, during incubation steps, reagents are leaking through the membrane by capillary action. As this liquid is generally not properly removed during the various washing steps, it will increase the back­ ground signal. To avoid this, we recommend that the plate bottom is peeled off at the end of step 9 of the procedure and that the reverse of the membrane is washed with running distilled water, as well as the regular washes with PBS– 0.05% Tween 20. 3. Automatization of the ELISPOT technique makes it suitable for use in clinical and hospital settings (Herr et al., 1997). The detection limit of the technique is between one and five cytokine-producing cells for a total of 105 cells, which makes it one of the most sensitive techniques for the detection of antigen-specific T cells. However, in contrast to the tetramer-MHC-class II technique, cytokine­ producing T cells are only detected following antigen-specific activation of the 468

Table 5. Human cytokine-specific (m)Abs for ELISpot Cytokine

Tracer (Phase) mAb

Catcher (m)Ab

IL-1β IL-2 IL-4 IL-5 IL-6 IL-10 IL-12p70 IFN-g TNF-a IL-17A IL-17F IL-17A/F

B-A15 B-G5 B-R14 B-Z25 B-E8 B-N10 B-T21 B-B1 B-F7 B-C49 B-G46 B-C49

B-Z8 Rabbit polyclonal Ab B-G28 Goat polyclonal Ab B-E4 B-T10 B-P24 B-G1 B-C7 B-B51 B-F60 B-F60

At present, the most complete, well-characterized set of (m)Abs for use in the ELISPOT assay is available from Diaclone, Besançon, France.

tttttt VII. BD™ CYTOMETRIC BEAD ARRAY (CBA)

A. Introduction Flow cytometry is a powerful analytical tool that enables the characterization of cells and subcellular organelles as well as particles (such as polystyrene beads) on the basis of size and granularity (light scatter characteristics) and a number of different para­ meters defined by fluorescent probes, including fluorescent antibodies and dyes (Shapiro, 1994, see also Chapter 1, Phenotyping and separation of leucocyte popula­ tions based on affinity labelling of Volume 32). Today, flow cytometry is widely applied to the development of multiplex sandwich immunoassays. These particlebased, flow cytometric immunoassays are capable of simultaneously identifying the types and measuring the levels of multiple soluble analytes within small samples of biological fluids. The broad dynamic range of fluorescent detection offered by flow cytometry and the efficient capturing of analytes by suspended particles enables these assays to use fewer sample dilutions and to obtain multiple sample measurements in a short time period. For these reasons, this technology provides an important tool for analysing the networks of biological response modifiers (BRMs) that are coexpressed by cells that mediate immune and inflammatory responses. Biological response modi­ fiers (BRMs), in particular cytokines, chemokines, and their receptors are popular target molecules for study (Chen et al., 1999; Cook et al., 2001; Funato et al., 2002). 469

Measuring Human Cytokine Responses

cells. Unless the cells are sorted before the assay, the ELISPOT method does not allow to phenotype the cytokine producing cells, neither to sort cytokine-pro­ ducing cells. A list of monoclonal and polyclonal Abs available for the detection of cytokines by ELISPOT is shown in Table 5.

The BDTM Cytometric Bead Array (CBA) from BD Biosciences employs a series of suspended beads to simultaneously detect and quantify multiple soluble analytes. Each bead has a unique fluorescence intensity so that they can be mixed and run simultaneously in a single tube to significantly reduce sample requirements and time to results in comparison with traditional ELISA and western blot techniques. BD CBA solutions are designed for multiplexed analysis providing more data using a single sample. Thus multiplexing is especially useful when only a small amount of sample is available, maximizing the number of proteins that can be analysed. With BD CBA, up to 30 proteins can be analysed with just 25–50 μl of sample. By compar­ ison, other methods such as ELISA and western blot require a similar amount of sample; however, only one protein can be analysed from the same volume.

B. Principle of the Assay BD CBA employs a series of different single size particles that are stably labelled with one (Kits) or two (Flex Sets) fluorescent dyes the emission wavelength of which is read at ~660 nm and over 680 nm. Each different group of beads is labelled with a discrete level of fluorescent dye so that it can be distinguished by its mean fluorescence intensity (MFI) upon flow cytometric analysis. For BD CBA Flex Sets, each bead population is given an alphanumeric position designation indicating its position relative to other beads (Figure 3). Beads with different positions can be combined to create a multiplex assay. In addition, beads within each group are covalently coupled with antibodies that can specifically capture a particular type of molecule present within biological fluids including sera, plasma, tears, tissue culture supernatants or cell lysates. By analogy with the ELISA method (pages 445), the antibody-coupled Capture Beads 105

NIR-A

104

103

102

102

103 Red-A

104

105

Figure 3. BD CBA 30-plex assay resolved on the BD FACSArray™ bioanalyser (Image from: BD CBA brochure (23-8910-01) p. 5).

470

Instrument

Reporter parameter

Clustering parameters for BD CBA flex sets

Clustering parameters for BD CBA kits

BD FACSArray™ bioanalyser BD FACSCanto™ II flow cytometer BD™ LSR II flow cytometer BD FACSAria™ II cell sorter BD FACSCalibur™ flow cytometer

Yellow PE PE PE FL2

Red and NIR APC and APC-Cy7 APC and APC-Cy7 APC and APC-Cy7 FL4 and FL3

Red APC APC APC FL3 or FL4

serve as the solid capture phase for the CBA. The immobilized, high-affinity antibodies function to specifically capture and localize analytes of interest that may be present in biological fluids tested. The captured analyte is then specifically detected by the addition of a fluorescent antibody. PE-coupled (emission at ~585 nm) detection antibodies are most frequently used since the emission wave­ length of PE is easily distinguishable from the emission wavelength(s) of the BD CBA capture beads. By including serial dilutions of a standard analyte solution (e.g. a mixture of cytokine protein standards with known concentrations), the BD CBA supports the development of standard curves for each analyte. With multi­ colour flow cytometric analysis, the levels of analytes captured by the different bead groups are distinguished by looking at the median fluorescence intensity (MFI) of the PE signal, which is in direct proportion to the amount of analyte present. The data are analysed using FCAP Array™ software to calculate the concentra­ tions of multiple analytes by plotting standard curves and calculating sample concentrations based on these curves. Due to the complexity of the BRM and cell signaling networks that underlie immune function, the capacity of the BD CBA to simultaneously measure multiple analytes in a single small-volume sample is highly advantageous. BD CBA technology has been optimized for digital flow cytometers but is compatible with virtually all dual-laser flow cytometers capable of detecting and distinguishing fluorescence emissions at 576, 670 and >680 nm (Table 6).

C. BD CBA Flex Sets 1. Introduction

The BD CBA Flex Set system provides an open and configurable menu of beadbased reagents designed to make it easy to create multiplex assays. Available assays include soluble protein assays for detection of human, mouse or rat cyto­ kines, chemokines and growth factors; human immunoglobulins; and cell signal­ ing assays for detection of phosphorylated cell signaling proteins. Using the BD 471

Measuring Human Cytokine Responses

Table 6. BD CBA Flex Sets instrument compatibility and associated parameters

CBA Flex Set system, up to 30 analytes can be measured simultaneously on a flow cytometer equipped with 488 and 633 nm lasers.

2. BD CBA Soluble Protein Flex Set assays for detection of cytokines

BD CBA Soluble Protein Flex Set assays are available for the detection of cytokines, chemokines, growth factors, and human Ig in serum, plasma or tissue culture supernatant samples. The antibody pair that comprises each assay is evaluated for dynamic range, sensitivity and parallel titration to native biological samples. The detection anti­ bodies are directly labelled with PE. By avoiding the streptavidin–biotin–PE detec­ tion method employed by other assays, direct PE detection reagents minimize the risk of increased background often caused by endogenous biotin in serum and lysate samples.

3. BD CBA Flex Set assay: step by step

BD CBA Flex Sets make it easy to build a multiplex by following three simple steps. First choose the analytes to be measured and the corresponding buffer kit. Then follow the simple formulation instructions in the Master Buffer Kit manual. Assay components are all formulated at 1 μl/test for easy calculations and the unique lyophilized standard pellets facilitate the combination of proteins to make the standards mix. The finished assay can be acquired on a variety of dual-laser flow cytometers and analysed using FCAP Array software. 1. Choose a human BD CBA Flex Set assay. Each BD CBA Flex Set comes with capture beads, detection reagent and standards. Sufficient reagents are provided to run 100 tests including two standard curves. All assays are available off-the-shelf and ready for mixing. 2. Choose a 100 or 500 test size BD CBA Flex Set Master Buffer Kit. Each BD CBA Flex Set Master Buffer Kit contains all the assay reagents and instrument setup beads necessary for any size multiplex configured from com­ patible BD CBA Flex Sets. This means that running a single-plex assay, a 10-plex assay or larger, the buffer reagents are optimized to perform with the custo­ mized mixture selected and yield the correct number of assay tests. 3. Perform the assay following the instructions in the Master Buffer Kit manual. 4. Acquire samples on a dual-laser flow cytometer. The BD CBA Flex Set reagents have been validated on a number of BD duallaser flow cytometry platforms (Table 6). The plate-based BD FACSArray bioa­ nalyser can be used to optimize assay throughput and workflow. Simply pre­ pare samples and standards in a 96-well filter-bottom plate, launch the previously designed FCAP Array template, load the plate and experience truly hands-free sample acquisition. 5. Analyse data files using FCAP Array multiplex analysis software. Use the intuitive, wizard-driven FCAP Array software to plot standard curves and calculate sample concentrations. 472

D. Notes and Recommandations 1. Quantitative results or protein levels for the same sample or recombinant protein run in ELISA and BD CBA assays may differ. A spike recovery assay can be performed using an ELISA standard followed by BD CBA analysis to assess possible differences in quantitation. So-called ‘Gold Standards’ further allow for comparing values obtained using different assay method with those of the National Institute for Biological Standards and Control (NIBSC)/World Health Organization (WHO) International Standards (page 454). 2. When several BD CBA Flex Sets assays are multiplexed, it is possible that the background (MFI of the 0 pg/mL standard point) may increase and the overall assay signals of other standard points may be reduced. This can result in lower dynamic range or loss in sensitivity in some assays. This effect may be greater as more assays are added to the multiplex. 3. For assays that will be acquired on a BD FACSCalibur flow cytometry instru­ ment, it is recommended that additional dilutions of the standard be prepared (i.e. 1:512 and 1:1024) as it is possible that in multiplex experiments containing a large number of assays, the Top Standard, 1:2 and 1:4 standard dilution will not be analysable by the FCAP Array software. In those cases, the Top Standard, 1:2 and 1:4 standard dilutions can be run on the experiment but may need to be excluded from the final analysis in the FCAP Array software (Cat. No. 641488).

Each BD CBA Flex includes specific Capture Beads, Detection Reagents and Stan­ dards. Assay buffers, Flow Cytometer Setup Reagents and Instruction Manual are provided in each BD CBA Flex Set Master Buffer kit. Instruction Manuals, Instru­ ment Setup instructions and Acquisition Templates can be downloaded at bdbiosciences.com/flexset In addition to the reagents provided in the BD CBA Master Buffer Kit and the BD CBA Flex Sets, a dual-laser flow cytometer equipped with a 488 or 532 nm laser, and a 633 or 635 nm laser and capable of distinguishing 576, 660 and >680 nm fluores­ cence is required for the assay. Refer to Table 6 for examples of compatible instru­ ment platforms. 1275 mm sample acquisition tubes for a flow cytometer (e.g., BD FalconTM Cat. No. 352008), and the FCAP Array software (Cat. No. 641488). 1. Required for running experiments on a BD FACSCalibur flow cytometer

• BD CaliBRITE™ 3 beads (Cat. No. 340486) • BD CaliBRITE APC beads (Cat. No. 340487) • BD FACSComp™ software 2. Required for plate loader-equipped flow cytometers

• Millipore MultiScreenHTS-BV 1.2 μm clear non-sterile filter plates (Cat. No. MSBVN1210 (10 pack) or MSBVN1250 (50 pack)) 473

Measuring Human Cytokine Responses

E. Material and Reagents

• Millipore MultiScreenHTS Vacuum Manifold (Cat. No. MSVMHTS00) • MTS 2/4 Digital Stirrer, IKA Works, VWR (Cat. No. 82006-096) • Standard microtitre plate for BD FACSArray Bioanalyzer Setup (BD Falcon Cat. No. 353910) • Vacuum source • Vacuum gauge and regulator (if not using recommended manifold)

F. BD CBA Soluble Protein Flex Set assay procedure 1. Overview

Summarized below is the BD CBA Flex Set assay procedure for measuring soluble proteins in human samples (see sections below for details). 1. 2. 3. 4. 5. 6. 7.

Perform instrument setup procedure. Dilute samples as appropriate. Reconstitute standards mix and prepare serial dilutions. Prepare (dilute) Capture Beads. Prepare (dilute) PE Detection Reagents. Transfer Capture Beads to tubes or wells. Add standard and sample dilutions to the appropriate tubes or wells. Incubate for 1 h at room temperature (protect from light). 8. Add mixed PE Detection Reagent to tubes or wells.

Incubate for 2 h at room temperature (protect from light).

9. Wash. 10. Analyse.

2. Instrument setup

In order to ensure that the flow cytometer is performing optimally, perform the instrument setup procedure prior to preparing the BD CBA Flex Set assay. Refer to the appropriate flow cytometry instrument setup in the manual included with the Master Buffer Kit for instructions on how to set up your instrument. 3. Preparation of test samples

The standard curve for each BD CBA Soluble Protein Flex Set covers a defined set of concentrations. It may be necessary to dilute test samples to ensure that their mean fluorescence values fall within the limits or range of the generated standard curve. For best results, samples that are known or assumed to contain high levels of a given protein should be diluted as described below. 1. Dilute test sample by the desired dilution factor (i.e. 1:10 or 1:100) using the appropriate volume of Assay Diluent. Serum or plasma samples must be diluted at least 1:4 before transferring the samples to the assay tubes or wells. 474

2. Mix sample dilutions thoroughly before transferring samples to the appropriate assay tubes containing Capture Beads. 3. In order to facilitate analysis in FCAP Array software, load serially diluted samples in sequential wells from most concentrated to least concentrated (e.g. Sample 1 – 1:4, 1:8, 1:16; Sample 2 – 1:4, 1:8, 1:16; etc.).

4. Preparation of BD CBA Soluble Protein Flex Set standards

1. Remove one lyophilized standard vial from each BD CBA Soluble Flex Set. 2. Open each vial of lyophilized standard and pool all lyophilized standard spheres into a single polypropylene tube (Recommended 15-ml Conical Tube, BD Falcon Cat. No. 352097). Label the tube ‘Top Standard’. 3. Reconstitute the standards with 4.0 ml of Assay Diluent. Allow the reconstituted standard to equilibrate for at least 15 min before making dilutions. Mix recon­ stituted protein by pipette only. Do not vortex or mix vigorously. 4. Label 12 × 75 mm tubes (BD Falcon Cat. No. 352008) and arrange them in the following order: 1:2, 1:4, 1:8, 1:16, 1:32, 1:64, 1:128 and 1:256. 5. Pipette 500 μl of Assay Diluent to each of the remaining tubes. 6. Perform a serial dilution by transferring 500 μl from the Top Standard to the 1:2 dilution tube and mix thoroughly. Continue making serial dilutions by transfer­ ring 500 μl from the 1:2 tube to the 1:4 tube and so on to the 1:256 tube and mix thoroughly. Mix by pipette only; do not vortex. Prepare one tube containing Assay Diluent to serve as the 0 pg/mL negative control (Table 7). 7. It is recommended that the first 10 wells or tubes in the experiment be the standards. Standards should be run in order from least concentrated (0 pg/mL) to most concentrated (Top Standard, see Table 8).

Table 7. Typical BD CBA Human Soluble Protein Flex Set standard concentrations after dilution BD CBA Human Soluble Protein Flex Set Standard Protein (pg/ml)

Top Standard

1:2 Dilution Tube

1:4 Dilution Tube

1:8 Dilution Tube

1:16 Dilution Tube

1:32 Dilution Tube

1:64 Dilution Tube

1:128 Dilution Tube

1:256 Dilution Tube

2500

1250

625

312.5

156

80

40

20

10

Note: Refer to the Technical Data Sheet for each individual assay to verify the concentration of the Top Standard. Table from: Manual for Cat. No. 558264, p. 11.

475

Measuring Human Cytokine Responses

For each single bead or multiplex assay, a standard curve will need to be prepared. The protocol below indicates how standards should be mixed and diluted for use in a BD CBA Soluble Flex Set assay.

Table 8. Essential controls Tube No.

Reagents (All reagent volumes are 50 μl)

1 2 3 4 5 6 7 8 9 10

Capture Beads, Assay Diluent, PE Detection Reagent Capture Beads, Standard 1:256 Dilution, PE Detection Reagent Capture Beads, Standard 1:128 Dilution, PE Detection Reagent Capture Beads, Standard 1:64 Dilution, PE Detection Reagent Capture Beads, Standard 1:32 Dilution, PE Detection Reagent Capture Beads, Standard 1:16 Dilution, PE Detection Reagent Capture Beads, Standard 1:8 Dilution, PE Detection Reagent Capture Beads, Standard 1:4 Dilution, PE Detection Reagent Capture Beads, Standard 1:2 Dilution, PE Detection Reagent Capture Beads, Standard ‘Top Standard’, PE Detection Reagent

Negative Control 10 pg/ml Standard 20 pg/ml Standard 40 pg/ml Standard 80 pg/ml Standard 156 pg/ml Standard 312.5 pg/ml Standard 625 pg/ml Standard 1250 pg/ml Standard 2500 pg/ml Standard

5. Preparation of BD CBA Soluble Protein Flex Set capture beads

The Capture Beads provided in each BD CBA Soluble Flex Set are at a 50× concentration and must be diluted to their optimal concentration before adding to a given assay tube or assay well. 1. Determine the number of BD CBA Soluble Protein Flex Sets to be used in the experiment (size of the multiplex). 2. Determine the number of tests in the experiment. It is recommended that the user prepare a few additional tests than they will use in the experiment to ensure that there is enough material prepared for the experiment. 3. Vortex each Capture Bead stock vial for at least 15 s to resuspend the beads thoroughly. 4. Determine the total volume of diluted beads needed for the experiment. Each tube/well requires 50 μl of the diluted beads. The total volume of diluted beads can be calculated by multiplying the number of tests (determined in step 2) by 50 μl. • e.g. 35 tests × 50 μl = 1750 μl total volume of beads 5. Determine the volume needed for each capture bead. Beads are supplied so that 1.0 μl = 1 test. Therefore, the required volume (μl) of beads is equal to the number of tests. • e.g. 35 tests require 35 μl of each capture bead included in the assay 6. Determine the volume of Capture Bead Diluent needed to dilute the beads. The volume of Capture Bead Diluent can be calculated by subtracting the volume for each bead tested from the total volume of diluted beads needed to perform the assay. • e.g. 1750 μl total volume of beads – 35 μl for each bead = volume of Capture Bead Diluent • e.g. if testing one analyte: 1750 μl – (35 μl × 1) = 1715 μl Diluent • e.g. if testing five analytes: 1750 μl – (35 μl × 5) = 1575 μl Diluent Refer to Table 9 for more examples of the calculation. 7. Pipette the Capture Beads and Capture Bead Diluent into a tube labelled Mixed Capture Beads. 476

Table 9. Capture bead and PE detection reagent diluent calculations

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30

1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

Total Capture Volume/ test (μl) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30

Volume of Capture Bead or Detection Reagent Diluent/ test (μl) 49 48 47 46 45 44 43 42 41 40 39 38 37 36 35 34 33 32 31 30 29 28 27 26 25 24 23 22 21 20

Total volume of mixed Capture Beads or PE Detection Reagents/ test (μl) 50 50 50 50 50 50 50 50 50 50

50

50

50

50

50

50

50

50

50

50

50

50

50

50

50

50

50

50

50

50

Table from: Manual for Cat. No. 558264, p. 20.

6. Notes and Recommendations

Due to some unspecific binding observed with human serum and plasma samples, a specific Capture Beads procedure has been implemented in order to avoid any interference with the assay. The Capture Bead Diluent for Serum/Plasma is included in the Human Soluble Master Buffer kit (Cat. Nos. 558264 and 558265). 1. Determine the number of BD CBA Human Soluble Protein Flex Sets to be used in the experiment (size of the multiplex). 2. Determine the number of tests in the experiment. It is recommended that the user prepare a few additional tests than they will use in the experiment to 477













Measuring Human Cytokine Responses

Number of Volume of each Flex Sets Capture Bead or PE to be used Detection Reagent/ test (μl)

3. 4. 5. 6. 7.

ensure that there is enough material prepared for the experiment. Beads are supplied so that 1.0 μl = 1 test. Therefore, the required volume (μl) of beads is equal to the number of tests. • e.g. 35 tests requires 35 μl of each capture bead included in the assay Vortex each Capture Bead stock vial for at least 15 s to resuspend the beads thoroughly. Pipette the appropriate volume (determined in step 2) of each capture bead into a tube labelled Mixed Capture Beads. Add 0.5 ml Wash buffer, centrifuge at 200 × g for 5 min. Carefully discard supernatant by aspiration. Avoid aspiration of the bead pellet. Resuspend beads in Capture Bead Diluent for Serum/Plasma to a final volume of 50 μl/test, vortex and incubate for 15 min at room temperature prior to use. • e.g. 35 tests × 50 μl = 1750 μl Capture Bead Diluent for Serum/Plasma

7. Preparation of BD CBA Soluble Protein Flex Set PE detection reagents

The PE Detection Reagent provided with each BD CBA Soluble Protein Flex Set is a 50× bulk (1 μl/test). The PE Detection Reagents for all BD CBA Protein Flex Sets used in the assay should be combined, and subsequently diluted to their optimal volume per test (50 μl/test) before adding the PE Detection Reagent Mix to a given tube or assay well. Note: Protect the PE Detection Reagents from exposure to direct light because they can become photobleached and will lose fluorescent intensity. 1. Determine the number of BD CBA Soluble Protein Flex Sets to be used in the experiment (size of the multiplex). 2. Determine the number of tests to be run in the experiment. It is recommended that the user prepare a few additional tests than they will use in the experiment to ensure that there is enough material prepared for the experiment. 3. Determine the total volume of diluted PE Detection Reagent needed for the experiment. Each tube/well requires 50 μl of the diluted PE Detection Reagent. The total volume of diluted PE can be calculated by multiplying the number of tests (determined above) by 50 μl. • e.g. 35 tests × 50 μl = 1750 μl total volume of PE 4. Determine the volume needed for each PE Detection Reagent. The PE Detection Reagent is supplied so that 1.0 μl = 1 test. Therefore, the required volume (μl) of PE Detection Reagent is equal to the number of tests. • e.g. 35 tests requires 35 μl of each Detection Reagent included in the assay 5. Determine the volume of Detection Reagent Diluent needed to dilute the PE Detection Reagents. The volume of Detection Reagent Diluent can be calculated by subtracting the volume for each PE Detection Reagent tested from the total volume of diluted PE needed. • e.g. 1750 μl total volume PE – 35 μl for each Detection Reagent = volume of Detection Reagent Diluent • e.g. if testing one analyte: 1750 μl – (35 μl × 1) = 1715 μl Diluent • e.g. if testing five analytes: 1750 μl – (35 μl × 5) = 1575 μl Diluent 478

Refer to Table 9 for more examples. 6. Pipette the Detection Reagents and Detection Reagent Diluent into a tube labelled Mixed PE Detection Reagents. Store at 4°C, protected from light until ready to use.

G. BD CBA Soluble Protein Flex Set assay procedure Following the preparation and dilution of the individual assay components, transfer the Standards or samples, mixed Capture Beads and mixed PE Detec­ tion Reagents to the appropriate assay wells or tubes for incubation and analysis. Note: Protect Capture Beads and PE Detection Reagents from direct exposure to light. 1. Overview

Samples

Capture Beads Mixture 50 µl 50 µl Diluted Standards 50 µl N/A Samples N/A 50 µl Vortex the tubes for 5 s or shake the plates for 5 min and incubate for 1 h at room temperature. PE Detection Reagent Mixture 50 µl 50 µl Mix assay tubes gently or shake the plates for 5 min and incubate for 2 h at room temperature in the dark. Apply the plate to the vacuum manifold and aspirate or add 1.0 ml of Wash Buffer to each assay tube, centrifuge at 200 × g for 5 min and discard the supernatant. Wash Buffer 150 µl for plates or 150 µl for plates or 300 µl for tubes 300 µl for tubes

2. Detailed protocol for plates or tubes

1. Prepare all reagents as described in previous sections before starting the experiment. 2. For plates: Pre-wet the plate by adding 100 μl of Wash Buffer to each well. To remove the excess volume, apply to vacuum manifold. Do not exceed 10″ Hg of vacuum pressure. Vacuum aspirate until wells are drained (2–10 s). 3. Vortex the Mixed Capture Beads for at least 5 s. Add 50 μl of the Mixed Capture Beads to each assay well (plates) or assay tube. 4. Add 50 μl of Standard or sample to the assay wells (plates) or each assay tube. 5. Mix the plate for 5 min using a digital shaker at 500 rpm (do not exceed 600 rpm) or genly mix the assay tubes and incubate for 1 h at room temperature. 479

Measuring Human Cytokine Responses

Standards

6. Add 50 μl of the Mixed PE Detection Reagent to each assay well (plates) or assay tube. 7. Mix the plate for 5 min using a digital shaker at 500 rpm or gently mix the assay tubes and incubate at room temperature for 2 h. 8a. For plates: Apply the plate to the vacuum manifold and vacuum aspirate (do not exceed 10″ Hg of vacuum pressure) until wells are drained (2–10 s). 8b. Add 150 μl of Wash Buffer to each assay well. Shake microwell plate on a digital shaker at 500 rpm for 5 min to resuspend beads. 9a. For tubes: Add 1.0 ml of Wash Buffer to each assay tube and centrifuge at 200 × g for 5 min. 9b. Carefully aspirate and discard the supernatant from each assay tube. 9c. Add 300 μl of Wash Buffer to each assay tube. Vortex assay tubes briefly to resuspend beads. 10. Analyse the samples on a flow cytometer. Proceed to the appropriate flow cytometry instrument instruction manual (included in the Master Buffer Kit) for acquiring the BD CBA Flex Sets. Note: It is best to analyse samples on the day of the experiment. Prolonged storage of samples, once the assay is complete, can lead to increased background and reduced sensitivity.

H. Cytometer setup, data acquisition and analysis Note: The BD CBA Instrument Setup Templates for different flow cytometers men­ tioned in this section can all be downloaded at: bdbiosciences.com/flexset 1. For flow cytometers running BD FACSDiva™ software

For optimal performance of a BD CBA assay, it is necessary to properly set up the flow cytometer. The cytometer setup information in this section is to be used for the BD FACSCanto™, BD FACSCanto II, BD™ LSR II, BD FACSAria™ and BD FACSAria II flow cytometers.

2. Preparation of instrument setup beads

Prepare five tubes labelled: A9, PE-F1, F1, F9 and A1. Vortex the stock vials of beads, add 200 μl of Wash Buffer to each tube followed by 25 μl of the correspond­ ing setup beads.

3. Instrument setup

1. Edit the parameters list to display only the following: FSC-A, FSC-W, SSC-A, SSC-W, PE-A, APC-A and APC-Cy7-A. 2. On a global worksheet, create the following plots: FSC-A/SSC-A dot plot, APC/ APC-Cy7 dot plot and PE histogram. 480

4. Verification of Instrument Setup

The BD CBA 30 Plex Bead Mixture (Cat. No. 558522) can be included as an additional sample when performing the instrument setup procedure. This cocktail containing 30 bead populations from the BD CBA Flex Set is useful as a control sample to demon­ strate that the instrument has been properly set up for analysis and to guarantee that the instrument and FCAP Array Software can analyse a 30 plex assay correctly. Recommended Assay Procedure: 1. Vortex the BD CBA 30 Plex Bead Mixture to bring beads into suspension. 2. Transfer 25 μl of the BD CBA 30 Plex Bead Mixture to the appropriate sample well or tube. 3. Add 175 μl of Wash Buffer (from the BD CBA Master Buffer Kit used) to the bead sample. More Wash Buffer (275 μl) may be needed for samples in tubes. 4. Proceed with analysis on a BD flow cytometer that has been set up according to the instrument setup procedure and export the FCS2.0 file for the 30 plex bead mixture. 5. Analyse the 30 plex bead mixture data file using the FCAP Array software clustering tool (see Technical Data Sheet for details). If the FCAP Array software fails to identify 30 bead clusters, repeat steps 4 and 5 after repeating the instrument setup procedure. If the FCAP Array software is unable to identify 30 bead clusters from the repeat sample, instrument service may be required. 481

Measuring Human Cytokine Responses

3. Set FSC-A and SSC-A to Log and create a statistics view showing the FSC-A and SSC-A means. Set the events to display to 500. Using the A9 setup beads, adjust FSC and SSC so that the singlet beads have a mean of 30,000 for each parameter. Stop acquisition to avoid running out of sample. 4. Adjust the FSC-A and SSC-A thresholds using the mean channel as a guideline. Be sure that the thresholds do not cut into the bead population. 5. In the FSC-A versus SSC-A dot plot, create a region that includes the singlet population of beads. In the Population Hierarchy, rename that region singlet. 6. Edit the statistics view to display the PE, APC and APC-Cy7 mean of the singlet beads. 7. Through the singlet gate, run the A9 setup beads and adjust the APC and APC­ Cy7 voltages until the mean of each parameter is 160,000 ± 2,000. 8. Through the singlet gate, run the PE-F1 tube and adjust the PE voltage so that the mean is 65 ± 5. 9. Create compensation controls and delete the PE compensation tube. Run beads as follows for compensation controls: • Unstained: F1 • APC Stained: F9 • APC-Cy7 Stained: A1 10. Calculate compensation. 11. Optional: Verify instrument settings prior to analysing the assay by recording a sample using the remaining mixed capture beads from the Flex Set assay, export as FCS2.0 and go to Tools > Clustering Test in FCAP Array software to see if it can identify the correct number of bead clusters.

Note: Do not store the diluted beads. Prepare a new bead sample for each test. The same procedure is recommended for verification of instrument setup when using the BD FACSArray bioanalyser. 5. Data acquisition

Set events to record to 300 events per analyte (e.g. 300 × 6 = 1800 events for a 6 plex) and set the singlet gate as the storage gate and stopping gate to ensure that only singlet bead events are recorded. Change events to display to 5000. Record samples and export as FCS2.0 files for analysis in FCAP Array. The Experiment can be saved as a template for future experiments; however, it is recommended to verify instrument settings (i.e. voltages and compensation) prior to each experiment. 6. Analysis of sample data

The analysis of BD CBA data is optimized when using FCAP Array software. Install the software according to the instructions in the FCAP Array User’s Guide (available for download at bdbiosciences.com/fcaparray). 1. Transfer FCS data files for the experiment to the computer with the FCAP Array software. 2. Place all data files for a given experiment in a single folder. Follow the instructions in the FCAP Array Software User’s Guide for creating an experiment and for data analysis.

I. For the BD FACSArray bioanalyser running BD FACSArray software For experiments requiring higher throughput, BD CBA reagents can optimally be combined with the BD FACSArray bioanalyser. Equipped with a 96-well plate loader, the BD FACSArray offers automated data acquisition. BD FACSArray soft­ ware is used for instrument setup and sample acquisition. BD FACSArray software is compatible with FCAP Array software to allow for quantitative analysis of BD CBA data. FCAP Array software enables researchers to design and set up their BD CBA experiments and import them to the BD FACSArray bioanalyser in a stream­ lined workflow. Refer to the BD FACSArray Bioanalyzer Instrument Setup Manual for instrument setup and data acquisition. Follow the instructions in the FCAP Array Software User’s Guide (available for download at bdbiosciences.com/fcaparray) for creating an experiment and for data analysis.

J. For the BD FACSCalibur™ flow cytometer For optimal performance of a BD CBA assay, it is necessary to properly set up the flow cytometer. The cytometer setup information in this section is to be used for the BD FACSCalibur™ flow cytometer. The BD FACSComp™ Software is useful for 482

setting up the flow cytometer. BD CellQuest™ (or BD CellQuest Pro) Software is required for analysing samples and formatting data for subsequent analysis using FCAP Array software.

1. Instrument setup with BD FACSComp software and BD Calibrite™ beads

1. 2. 3. 4. 5.

Perform instrument startup. Perform flow check. Prepare tubes of BD Calibrite™ beads for a four-colour setup. Launch BD FACSComp software. Run BD FACSComp software in Lyse/No Wash mode. Note: Time-delay calibration must be performed. For detailed information on using BD FACSComp with BD Calibrite beads to set up the flow cytometer, refer to the BD FACSComp Software User’s Guide and the BD Calibrite Beads package insert. 6. Proceed to the next section.

1. Label five 12 × 75 mm tubes A, B, C, D and E. 2. Add 25 μl of PE Instrument Setup Bead F1 to tube D. 3. Add 50 μl of PE Positive Control Detector to tube D and vortex tube briefly to mix. 4. Incubate tube D for 15 min at room temperature, protect from light. 5. Add 25 μl of Instrument Setup Bead A9 to tube A. 6. Add 25 μl of Instrument Setup Bead A1 to tube B. 7. Add 25 μl of Instrument Setup Bead F9 to tube C. 8. Add 25 μl of Instrument Setup Bead F1 to tubes A, B, C and D. 9. Add 50 μl of the mixed Capture Beads from the BD CBA Flex Set experiment to tube E. Note: The mixed Capture Beads added in step 9 should be beads only and not contain PE detection reagent, standards or sample.

Tube

Setup beads

A B C D E

A9 + F1 A1 + F1 F9 + F1 PE-F1 + F1 Mixed capture beads

10. Add 300 μl of Wash Buffer to tube D and vortex tube briefly to mix. 11. Add 350 μl of Wash Buffer to tubes A, B, C and E. Vortex each tube briefly to mix. 12. Proceed to the next section. 483

Measuring Human Cytokine Responses

2. Preparation of instrument setup beads

3. Instrument setup with instrument setup beads

1. Launch BD CellQuest (or BD CellQuest Pro) software and open the BD CBA Flex Set Setup Template. Note: The BD CBA Flex Set Setup Template file can be downloaded at bdbiosciences. com/flexset 2. Set the instrument to Acquisition mode. 3. Set SSC and FSC to Log mode. 4. Decrease the SSC PMT voltage by 100 from what BD FACSComp set. 5. Set the Threshold to SSC at 650. 6. In Setup mode, run tube A. Follow the setup instructions on the following pages. Note: Pause and restart acquisition frequently during the instrument setup procedure to reset detected values after settings adjustments. Adjust gate R1 so that the singlet bead population is located in gate R1 (Figure 4). Adjust the R2 gate so it gates the brightest bead population (Figure 5). Adjust the FL3 PMT so that the FL3 median of the A9 bead population is around 1500 and then adjust the FL4 PMT so that the FL4 median of the A9 bead population is around 5000. 120804hs.001

104

R1

SSC-H

103 102 101 100 100

101

102

103

104

FSC-H

Figure 4. Instrument setup procedure. 120804hs.001

104

R2 FL3 Median: 1512.47

FL3-H

103 102 101

FL4 Median: 5048.07

100 100

101

102 FL4-H

Figure 5. Instrument setup procedure.

484

103

104

Adjust gate R3 so the F1 bead population is inside it (Figure 6). Adjust the FL2 PMT so the FL2 median of the F1 bead population is between 2.5 and 4.0. Proceed to the next page of the setup template. Run tube B to adjust the compensation settings for FL4 – %FL3. Adjust gate R4 so the A1 bead population (brightest bead in FL3 channel) is inside it (Figure 7). Ensure that the left edge of gate R4 is touching the y-axis boundary of the dot plot. Adjust gate R5 so the F1 bead population is inside it (Figure 7). Using the FL4 – %FL3 control, adjust the median of G4 until it is equal to the median of G5. Proceed to the next page of the setup template. Run tube C to adjust the compensation settings for FL3 – %FL4. 120804hs.001

104

103 FL3-H

FL2 Median: 4.0 102 R3 101

100 101

102 FL2-H

103

104

Measuring Human Cytokine Responses

100

Figure 6. Instrument setup procedure.

120804hs.002

104

R4

FL3-H

103

Gate G4 Median: 12.30

102

Gate G5 Median: 12.19

R5 101

100 100

101

102 FL4-H

Figure 7. Instrument setup procedure.

485

103

104

Adjust gate R6 so the F1 bead population (dimmest bead in FL4 channel) is inside it (Figure 8). Adjust gate R7 so the F9 bead population is inside it. Using the FL3 – %FL4 control, adjust the median of G7 until it is equal to the median of G6. Proceed to the next page of the setup template. Run tube D to adjust the compensation settings for FL3 – %FL2. Adjust gate R8 so the F1 bead population (dimmest bead in FL2 channel) is inside it (Figure 9). Adjust gate R9 so the PE-F1 bead population is inside it. Using the FL3 – %FL2 control, adjust the median of G9 until it is equal to the median of G8. Proceed to the next page of the setup template. 120804hs.003

104

103

R7

FL3-H

R6 Gate G6 Median: 54.25

102

Gate G7 Median: 50.48

101

100 100

101

102 FL4-H

103

104

Figure 8. Instrument setup procedure.

120804hs.004

104

103

FL3-H

R8

R9 Gate G8 Median: 72.34

102

Gate G9 Median: 72.34 101

100 100

101

102 FL2-H

Figure 9. Instrument setup procedure.

486

103

104

120804hs.003

104

R10 103

R11

Gate G10 Median: 2.81

R12

Gate G11 Median: 5.19

FL3-H

R13 102

R14

Gate G12 Median: 4.96 Gate G13 Median: 5.05 Gate G14 Median: 3.75

101

100 100

101

102 FL2-H

103

104

Run tube E to adjust the compensation settings for FL2 – %FL3. Adjust gates R10, R11, R12, R13 and R14 so each bead row falls within only one gate (see Figure 10). Ensure the left edge of each gate is touching the y-axis boundary of the dot plot. Using the FL2 – %FL3 control, adjust until the median of any row (G10, G11, G12, G13 or G14) equals 2 – 4.

K. Typical Sample data Note: It is important not to overcompensate the bead populations in this step. No bead population should have a median below 2. Note: Not all populations shown in Figure 10 will be displayed in every setup; the populations displayed are dependent on the Flex Set beads used in the experiment. 4. Data acquisition

1. Return to page 1 on the BD CBA Flex Set Setup Template. 2. Set the instrument to Acquisition mode. 3. In the Acquisition and Storage window, do the following: a. Set the Acquisition Gate to Accept G1 = R1 events. (This will allow for only the events that fall into R1 to be saved.) Note: Be sure that R1 is set correctly to avoid data loss. b. Set Collection Criteria for acquisition to stop at 300 × the number BD CBA Flex Set assays being used (e.g. 300 × 6 = 1800 events for a 6 plex). This 487

Measuring Human Cytokine Responses

Figure 10. Instrument setup procedure.

Human PBMCs 1000000

100000

Concentration (pg/mL)

10000

1000

100

IgE

MIG

IL-13

IFN-γ

IL-12p70

TNF

MIP-1β

LT-α

MCP-1

IL-3

RANTES

G-CSF

GM-CSF

FasL

Eotaxin

bFGF

Angiogenin

VEGF

IL-9

IL-10

IP-10

IL-8

MIP-1α

PMA/Ionomycin anti-CD3/CD28

IL-1β

IL-7

IL-6

IL-5

IL-4

IL-2

1

OSM

10

Figure 11. Human peripheral blood mononuclear cells (PBMCs) were stimulated under two different conditions. Supernatants were collected and measured in a BD CBA Flex Set assay (30 plex).

ensures that the sample file contains approximately 300 events of each bead population. Do not acquire more than 300 beads per population. c. Set Resolution to 1024. d. Click OK. 4. In setup mode, run tube number 1 and using the FSC versus SSC dot plot, place the R1 region gate around the singlet bead population (see Figure 4). 5. Samples are now ready to be acquired. 6. Begin sample acquisition with the flow rate set to LOW. Using the lowest flow rate can improve resolution of the individual bead populations in the bead plex. To facilitate analysis of data files using FCAP Array software and to avoid confu­ sion, add a numeric suffix to each file that corresponds to the assay tube number (i.e. Tube No. 1 containing 0 pg/ml could be saved as KT032598.001). The file name must be alphanumeric (i.e. contain at least one letter). 5. Analysis of sample data

The analysis of BD CBA data is optimized when using FCAP Array software. Install the software according to the instructions in the FCAP Array User’s Guide. 1. Transfer FCS data files for the experiment to the computer with the FCAP Array software. 2. Place all data files for a given experiment in a single folder. Follow the instructions for creating an experiment and data analysis in the FCAP Array Software User’s Guide. 488

Median Fluorescence (MFI)

10,000

1000 Human CBA Standard

100

NIBSC/WHO Iternational Standard 10

1 10

100 1000 Concentration

10,000

Figure 12. Titration curve comparing the BD CBA Human IFN-g recombinant standard to the NIBSC/WHO International standard (Image from: BD CBA brochure (23-8910-01) p. 12).

Laboratories throughout the world use different assays to measure soluble pro­ teins in biological samples. To support the comparison of values obtained with the BD CBA method, we have evaluated the performance of many of our BD CBA standards with gold standards from the NIBSC. The NIBSC protein standards are recognized by the WHO as international biological standards. They meet estab­ lished requirements for accuracy, consistency and stability. The NIBSC/WHO standards are assigned potency values in International Units (IU) of biological activity and nominal mass (i.e. not absolute mass values); therefore, they cannot be used to establish absolute concentrations for a cytokine preparation. However, these standards do provide a means to facilitate comparisons of cytokine concen­ tration values determined by experiments conducted within different laboratories or methods. The source of a recombinant protein (i.e. insect cell, E. coli, etc.) and the affinity of antibodies used can affect the measurement and performance of a protein in an immunoassay. The conversion factors provided in Tables 10 and 11 make it possible to compare protein concentrations present in samples measured by different immunoassays that have been standardized to the same NIBSC/WHO standards. The conversion factor may change based on the batch of either standard. Therefore, the conversion factor is intended to be a guideline indicating whether a BD CBA assay over- or underestimates analyte concentrations rela­ tive to the NIBSC/WHO standards. Researchers are advised to incorporate both sets of standards in their assays if they wish to derive data from the NIBSC/ WHO standards.

489

Measuring Human Cytokine Responses

L. Standardization of BD CBA standards to the NIBSC/WHO international standards

Table 10. NIBSC conversion factor summary for BD CBA Flex Set standards NIBSC Standard Code Mass Number units/ vial Human b-FGF Human G-CSF Human GM-CSF Human IFN-g Human 1L-1β Human IL-2 Human IL-3 Human IL-4 Human IL-5 Human 1L-6 Human IL-7 Human 1L-8 Human IL-9 Human IL-10 Human IL-11 Human IL-12p70 Human IL-13 Human MCP-1 Human MlP-la Human OSM Human RANTES

90-712 88-502 88-646 87-586 86-680 86-504 91-510 88-656 90-586 89-548 90-530 89-520 91-678 93-722 92-788 95-544 95-622 92-794 92-518 93-564 92-520

Human TNF Human TNFRI Human TNFRII Human VEGF Mouse GM-CSF

88-786 96-528 93-524 02-286 91-658

Mouse IL-1β

93-668

Mouse IL-2 Mouse IL-3

93-566 91-662

Mouse IL-4

91-656

Mouse IL-6

93-730

Mouse TNF

88-532

IU Value

4 μg 100 ng 1 μg 12.5 ng 1 μg 7.6 ng 1 μg 100 ng 500 ng 1 μg 1 μg 1 μg 100 ng 1 μg 500 ng 1 μg 1 μg 5 μg 2 μg 1 μg 10 μg

1600 10,000 10,000 250 100,000 100 1700 1000 5000 100,000 100,000 1000 1000 5000 5000 10,000 1000 5000 arbitrary units 200 arbitrary units 25,000 100,000 arbitrary units 46,500 1 μg 10 μg not provided 10 μg not provided 13 μg 13,000 100,000 arbitrary 1 μg units 100 ng 100,000 arbitrary units 100 ng 10,000 arbitrary 100,000 arbitrary 1 μg units 10,000 arbitrary 1 μg units 100 ng 10,000 arbitrary units 200,000 arbitrary 1 μg units

Table from: BD CBA brochure (23-8910-01) p. 13.

490

Calculated Concentration using BD CBA Flex Set (pg/ml)

Nominal NIBSC Concen­ tration (pg/ml)

BD CBA Flex Set: NIBSC/WHO mass conversion factor

1295.87 ± 103.20 1231.33 ± 87.71 1551.45 ± 70.33 1287.44 ± 174.12 2610.76 ± 129.84 2148.20 ± 193.43 1822.31 ± 117.76 1535.99 ± 79.24 1084.09 ± 254.01 2041.22 ± 220.02 573.00 ± 94.55 2448.42 ± 153.34 601.58 ± 35.24 2510.49 ± 144.61 13,401.31 ± 1390.37 1307.09 ± 71.6 2723.24 ± 660.88 2280.63 ± 150.6 1242.93 ± 40.98 531.11 ± 34.17 1589.05 ± 181.79

2500 2500 2500 2500 2500 2500 2500 2500 2500 2500 2500 2500 2500 2500 10,000 2500 2500 2500 2500 2500 2500

1.97 2.06 1.62 1.97 0.97 1.17 1.39 1.65 2.38 1.25 4.44 1.04 4.20 1.01 0.75 1.93 0.93 1.10 2.04 4.75 1.60

1642.65 ± 99.14 12,214.45 ± 1165.96 707.95 ± 46.45 1250.615 ± 72.77 1748.55 ± 132.95

2500 10,000 2500 2500 2500

1.54 0.83 3.57 2.03 1.46

4531.11 ± 393.89

2500

0.56

368.74 ± 41.31 1309.44 ± 77.95

2500 2500

6.92 1.93

439.64 ± 43.03

2500

5.76

221.70 ± 49.24

2500

11.40

1540.95 ± 191.70

2500

1.67

Table 11. NIBSC conversion factor summary for BD CBA kit standards

Human IFN-g Human 1L-1β Human IL-2 Human IL-4 Human IL-5 Human 1L-6 Human 1L-8 Human IL-10 Human IL-12p70 Human MCP-1

Code Mass Number units/ vial

IU Value

Calculated Concentration using BD CBA Kit (pg/ml)

87-586 86-680 86-504 88-656 90-586 89-548 89-520 93-722 95-544

12.5 ng 1 μg 7.6 ng 100 ng 500 ng 1 μg 1 μg 1 μg 1 μg

250 100,000 100 1000 5000 100,000 1000 5000 10,000

4495.12 ± 657.67 3463.92 ± 184.87 3118.99 ± 449.33 5717.84 ± 632.51 3892.01 ± 501.66 4986.28 ± 633.36 3359.12 ± 319.33 4389.72 ± 469.84 3764.63 ± 276.73

5000 5000 5000 5000 5000 5000 5000 5000 5000

1.12 1.46 1.61 0.88 1.30 1.01 0.74 1.14 1.34

92-794

5 μg

2653.49 ± 159.89

5000

0.95

1116.63 ± 90.04

5000

2.26

3651.85 ± 417.15 627.37 ± 26.29

5000 5000

1.38 8.03

867.48 ± 59.91

5000

5.81

486.43 ± 52.42

5000

10.26

2328.01 ± 140.83

5000

2.17

Human RANTES

92-520

Human TNF Mouse IL-2

88-786 93-566

Mouse IL-4

91-656

Mouse IL-6

93-730

Mouse TNF

88-532

5000 arbitrary units 10 μg 100,000 arbitrary units 46,500 1 μg 100 ng 10,000 arbitrary units 10,000 1 μg arbitrary units 100 ng 10,000 arbitrary units 200,000 1 μg arbitrary units

Nominal NIBSC BD CBA Kit: NIBSC/ Concen-tration WHO mass (pg/ml) conversion factor

Table from: BD CBA brochure (23-8910-01) p. 14.

Acknowledgements The authors would like to thank Ulf Andersson (Karolinska Hospital, Stockholm, Sweden) and Eric Tartour (Hôpital Européen Georges Pompidou, Paris, France) for sharing information. Schering-Plough Biopharma is supported by Schering-Plough Corporation.

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NIBSC Standard

Assenmacher, M., Schmitz, J. and Radbruch, A. (1994). Flow cytometric determination of cytokines in activated murine T helper lymphocytes: expression of interleukin-10 in interferon-g and in interleukin-4-expressing cells. Eur. J. Immunol. 24, 1097–1101. Chen, R., Lowe, L., Wilson, J. D., Crowther, E., Tzeggai, K., Bishop, J. E. and Varro, R. (1999). Simultaneous quantification of six human cytokines in a single sample using microparticlebased flow cytometric technology. Clin. Chem. 45, 1693–1694. Cook, E. B., Stahl, J. L., Lowe, C., Morgan, L. R., Wilson, E. J., Varro, R., Chan, A., Graziano, F. M. and Barney, N. P. (2001). Simultaneous measurement of six cytokines in a single sample of human tears using microparticle-based flow cytometry: allergics vs. non-allergics. J. Immunol. Methods 254, 109–118. Czerkinsky, C., Andersson, G., Ekre, H. P., Nilsson, L.-A., Klareskog, L. and Ouchterlony, Ö. (1988). Reverse ELISPOT assay for clonal analysis of cytokine production. I. Enumeration of g-interferon-secreting cells. J. Immunol. Methods 110, 29–36. Czerkinsky, C., Nilsson, L.-A., Nygren, H., Ouchterlony, Ö and Tarkowski, A. (1983). A solid-phase enzyme-linked immunospot (ELISPOT) assay for enumeration of specific Ab-secreting cells. J. Immunol. Methods 65, 109. Didenko, V. V. (2001). DNA probes using fluorescence resonance transfer (FRET): designs and Applications. Biotechniques 31, 1106–1121. Ferre, F. (1992). Quantitative or semi-quantitative PCR: reality versus myth. PCR Methods Appl. 2, 1–9. Funato, Y., Baumhover, H., Grantham-Wright, D., Wilson, J., Ernst, D. and Sepulveda, H. (2002). Simultaneous measurement of six human cytokines using the Cytometric Bead Array System, a multiparameter immunoassay system for flow cytometry. Cytometry Res. 12, 93–99. Gilliland, G., Perrin, S., Blanchard, K. and Bunn, H. F. (1990). Analysis of cytokine mRNA and DNA: detection and quantitation by competitive polymerase chain reaction. Proc. Natl. Acad. Sci. USA 87, 2725–2729. Herr, W., Linn, B., Leister, N., Wandel, E., Meyer zum Buschenfelde, K. H. and Wolfel, T. (1997). The use of computer-assisted video image analysis for the quantification of CD8+ T lymphocytes producing tumor necrosis factor a spots in response to peptide antigens. J. Immunol. Methods 203, 141–152. Higuchi, R., Dollinger, G., Walsh, P. S. and Griffith, R. (1992). Simultaneous amplification and detection of specific DNA sequences. Biotechnology 10, 413–417. Higuchi, R., Fockler, C., Dollinger, G. and Watson, R. (1993). Kinetic PCR: real-time monitoring of DNA amplification reactions. Biotechnology 11, 1026–1030. Holland, P. M., Abramson, R. D., Watson, R. and Gelfand, D. H. (1991). Detection of specific polymerase chain reaction product by utilizing the 5’ to 3’ exonuclease activity of Thermus aquaticus DNA polymerase. Proc. Natl. Acad. Sci. USA 88, 7276–7280. Ihle, J. N. (2001). The Stat family in cytokine signaling. Curr. Opin. Cell Biol. 13, 211–217. Jung, T., Schauer, U., Heusser, C., Neumann, C. and Rieger, C. (1993). Detection of intracellular cytokines by flow cytometry. J. Immunol. Methods 159, 197–207. Korn, T., Bettelli, E., Oukka, M. and Kuchroo, V.K. (2009). IL-17 and Th17 Cells. Ann Rev Immunol 27, 485–517. Novick, D., Kim, S. H., Fantuzzi, G., Reznikov, L. L., Dinarello, C. A. and Rubinstein, M. (1999). Interleukin-18 binding protein: a novel modulator of the Th1 cytokine response. Immunity 10, 127–136. Openshaw, P., Murphy, E. E., Hosken, N. A., Maino, V., Davis, K., Murphy, K. and O’Garra, A. (1995). Heterogeneity of intracellular cytokine synthesis at the single-cell level in polarized T helper 1 and T helper 2 populations. J. Exp. Med. 182, 1–11.

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Addendum: List of Manufacturers Refered to in This Chapter AELVIS Automated Elisa-spot assay videeo analysis systems

http://www.aelvis.net/index.php

http://www.atcc.org Hybridomas and antibodies for ELISA, ELISPOT and intracellular staining Amersham/Pharmacia-General Electric http://www.apbiotech.com Columns for primer and probe purification for use in real-time PCR Applied Biosystems http://home.appliedbiosystems.com

Reagents for quantitative RT-PCR

BD Biosciences http://www.bdbiosciences.com (m)Abs for cellular activation, intracellular staining, BD CBA, flowcytometer equipment BD Pharmingen PharMingen http://www.bdbiosciences.com (m)Abs for cellular activation, ELISA, ELISPOT and intracellular staining Biosource International http://www.biosource.com

Reagents for quantitative RT-PCR

493

Measuring Human Cytokine Responses

American Type Culture Collection

Calbiochem-Novabiochem http://www.calbiochem.com Reagents for T cell activation Caltag http://www.caltag.com Fluorochrome-conjugated antibodies Carl Zeiss http://www.zeiss.de ELISPOT detection system Clontech http://www.clontech.com Reagents for RT-PCR CTL-Cellular Technologies LTD ELISPOT Technology and reagents http://www.immunospot.com/ DAKO http://www.dako.com antibodies for ELISPOT assay Diaclone Research-Genprobe http://www.diaclone.com (m)Abs for cellular activation, ELISA, ELISPOT and intracellular staining Dynatech Corporation http://www.dynatech.com/html Elisa plates and spectrophotometer equipment Epicentre Technologies http://www.epicentre.com Brefeldin A Eppendorf http://www.eppendorf.com PCR machines, multipipetor equipment Intergen Company http://www.intergen.com Reagents for quantitative RT-PCR Invitrogen (Gibco) http://www.invitrogen.com Reagents for RT-PCR MABTECH Reagents for ELISPOT assay http://www.mabtech.com/ Millipore http://www.millipore.com ELISPOT plates

494

Molecular Devices http://www.moleculardevices.com Equipment for ELISA, software Molecular Dynamics http://www.mdyn.com Scanning equipment Molecular Probes http://www.probes.com Reagents for RT-PCR Moss Substrates Inc. http://www.MossSubstrates.com Substrates for ELISA Perkin-Elmer http://www.perkinelmer.com PCR machines, reagents for quantitative RT-PCR, software for primer sequence detemination Promega http://www.promega.com Reagents for RT-PCR

Quantum-Appligene http://www.quantum-appligene.com Products for RNA isolation Qiagen http://www.qiagen.com Reagents for RT-PCR, Reagents for quantitative RT-PCR R&D Systems http://www.rndsystems.com mAbs for cellular activation, ELISA and intracellular staining Roche http://www.roche.com PCR machines, reagents for quantitative RT-PCR Sigma-Genosys http://www.genosys.com Reagents for quantitative RT-PCR Southern Biotechnology Associates http://southernbiotech.com Enzymes and substrates for ELISA

495

Measuring Human Cytokine Responses

Prozyme http://www.prozyme.com Enzymes and substrates for ELISA

Stratagene http://www.stratagene.com PCR machines, Reagents for quantitative RT-PCR, Molecular Beacons Vector Laboratories http://www.vectorlabs.com Fluorochrome-conjugated antibodies

496