[29] Fluorophore-assisted light inactivation for multiplex analysis of protein function in cellular processes

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[29] Fluorophore-Assisted Light Inactivation for Multiplex Analysis of Protein Function in Cellular Processes B y BRENDA K. EUSTACE a n d DANIEL G. JAY

Introduction The application of photons to the imaging and analysis of biological processes has made great strides. This includes the ability to observe single-molecule interactions and advances in light microscopy using green fluorescent protein (GFP) fusions and fluorescence energy transfer to show dynamic protein interactions in cells. Photons are also used to move or hold microscopic objects and to measure nanoscale forces using optical tweezers. One aspect of biophotonics that is less developed is the use of photons as a means to manipulate in situ function. Photodynamic therapy, the use of photosensitizing reagents in conjunction with light, has been applied to kill specific cells, for example, in cancer treatment. 1 We and others have extended this approach by developing chromophore-assisted laser inactivation (CALl). CALl is a method to disrupt protein function in cellular context2 as a means to address the roles that specific proteins play in cells and model organisms. 3'4 Since its invention CALl has been applied to address in situ function for many proteins in a wide variety of cellular processes. CALl has thus far largely been applied in a hypothesis-driven manner, assessing one protein at a time. To take a more discovery-based approach, it is necessary to perform this functional inactivation in a high-throughput manner. For this purpose, we have developed fluorophore-assisted light inactivation (FALl). 5 FALl can be performed with diffuse light, such that many samples may be done in parallel using multiwell plates. This provides an unprecedented throughput in the ability to directly address the functional roles of specific proteins in cellular assays. This innovation allows for more global applications of light inactivation such that a functional proteomic approach to biological research is possible. In this article we describe CALl and FALl and their applications and methods. We include the set-up of light sources and labeling of reagents with isothiocyanate derivatives of malachite green and fluorescein. We close by describing methods to show how FALl may be applied

I L. Dalla Via and S. Marciani Magno, Curr. Med. Chem. 8, 1405 (2001). 2 D. G. Jay, Proc. Natl. Acad. Sci. U.S.A. 85, 5454 (1988). 3 A. E. Beermann and D. G. Jay, Methods Cell Biol. 44, 715 (1994). 4 E. W. Wong and D. G. Jay, Methods EnzymoL 325, 482 (2000). 5 S. Beck, T. Sakurai, B. Eustace, G. Beste, R. Schier, E Rudert, and D. G. Jay, Proteomics 2, 247 (2002).

METHODSIN ENZYMOLOGY,VOL.360

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in a multiplex fashion in a particular cellular assay for cancer cell migration and invasion. Advances in genomics and proteomics have provided a new challenge for biologists: assigning functional roles to the enormous number of proteins in the cell. It is estimated that there are 30,000 genes in the human genome. These genes translate into about 1 million different protein products generated by pre- and posttranslational modifications that can each confer a unique function to the protein. One common approach to address in situ protein function is to use functionblocking antibodies, and these reagents have been used to identify important proteins in many cellular processes. 6-8 Function-blocking antibodies have a significant benefit because they may be used directly for therapeutics. 9 Unfortunately, only a small proportion (1-5%) of antibodies raised against proteins of interest is able to effectively and specifically block function. 1° Because the vast majority of antibodies directed to particular proteins do not block function, we developed CALl to utilize these reagents to specifically inactivate proteins (Fig. 1A). 2 The non-function-blocking antibodies are multiply conjugated with a chromophore, malachite green (MG). When these antibodies are irradiated with high-powered pulsed laser light at a wavelength of 620 nm (absorbance of MG), short-lived hydroxyl radicals are formed. These short-lived radicals generated during CALl generate a half-maximal radius of damage of 15/~,11 causing oxidative damage to the bound antigen while neighboring proteins and subunits of the same protein are largely unaffected. This distance is compatible with the use of CALl in cells because the average intermolecular distance between proteins in cells is ~80/~. This specificity was illustrated with the T cell receptor complex, in which one subunit could be inactivated in T cells while other associated subunits were functionally undamaged during CALI. 12 CALl has been used to study a diverse array of different proteins, including membrane proteins, signaling molecules, cytoskeletal elements, and transcription factors (Table I). Micro-CALI, a related approach in which the laser is focused to micron diameters by microscope optics, has been used extensively to assess protein function in single cells. 13,14 Micro-CALl has been reviewed elsewhere, and is thus not 6 D. M. O'Rourke and M. I. Greene, lmmunol. Res. 17, 179 (1998). 7 j. Baselga, Ann. OncoL 11, 187 (2000). 8 H. Hashida, A. Takabayashi, M. Adachi, T. Imai, K. Kondo, N. Kohno, Y. Yamaoka, and M. Miyake, Int. J. Oncol. 18, 89 (2001). 9 Z. Fan and J. Mendelsohn, Curr. Opin. Oncol. 10, 67 (1998). 10 B. K. Muller and F. Bonhoeffer, Curr. BioL 5, 1255 (1995). 11 j. C. Liao, J. Roider, and D. G. Jay, Proc. Natl. Acad. Sci. U.S.A. 91, 2659 (1994). 12 j. C. Liao, L. J. Berg, and D. G. Jay, Photochem. Photobiol. 62, 923 (1995). 13 H. Y. Chang, K. Takei, A. M. Sydor, T. Born, E Rusnak, and D. G. Jay, Nature (London) 376, 686 (1995). 14 p. Diamond, A. Mallavarapu, J. Schnipper, J. Booth, L. Park, T. P. O'Connor, and D. G. Jay, Neuron 11, 409 (1993).

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A

OH

4

4'° OH m

102~102 • tO

FIG. 1. Comparison of the mechanisms of CALl and FALl. (A) A non-function-blocking antibody is labeled with the chromophore malachite green (MG). This antibody binds to the protein and on excitation of MG by 620-nm pulsed laser light, hydroxyl radicals are generated. These free radicals specifically modify the protein bound to the antibody without affecting neighboring proteins. (B) A nonfunction-blocking antibody is labeled with the fluorophore fluorescein. This antibody binds to the protein and, on excitation of fluorescein by diffuse light (fluorescein excitation, 494 nm), singlet oxygen molecules are generated. These singlet oxygen molecules specifically modify the protein bound to the antibody without affecting neighboring proteins. discussed here. 4'15 C A L l is an effective technique to directly address protein function in cells and a high-throughput C A L l approach w o u l d be beneficial for multiplex applications. To develop high-throughput applications, we have investigated an alternative a p p r o a c h to inactivate proteins in cells using diffuse light.

15A. Buchstaller and D. G. Jay, Microsc. Res. Tech. 48, 97 (2000).

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TABLE I PROTEINS INACTIVATEDBY CHROMOPHORE-ASSISTED LASER INACTIVATION Class and protein

ReL

Enzymes /~-Galactosidase (in vitro) Alkaline phosphatase (in vitro) Acetylcholinesterase (in vitro) Caspase 3 (in vitro and in cells)

2 2 2 17'~

Signal transduction molecules Calcineurin (in vitro and in cells) IP3 receptor (in vitro and in cells) MAP kinase (in vitro)

13 29 a 17a

Surface proteins Grasshopper fasciclin I (in vivo) Grasshopper fasciclin II (in vivo) c~ Chain of T cell receptor (in cells) fl Chain of T cell receptor (in cells) e Chain of T cell receptor (in cells) Drosophila patched protein (in vivo, mimicks hypomorphic mutation) NCAM (in vitro and in cells) L1 (in vitro and in cells) RGM (in vitro) FMRF amide receptor (in vitro) Fas receptor (in cells) fll-Integrin (in cells) Transcription factors Drosophila even skipped (in vivo, mimicks genetic loss of function) Tribolium even skipped (in vivo)

31 14 12 12 12 32 33 33 10 34a 176 5 35 36

Proteins not inactivated by CALl Hexokinase Glyceraldehyde-3-phosphate dehydrogenase Cytoskeletal proteins Myosin V (in vitro and in cells) Talin (in ceils) Radixin (in cells) Kinesin (in vitro) Hamartin (TSC I) (in ceils) Tau (in vitro and in cells) Myosin 1/~ (in cells) Vinculin (in vitro and in cells) Ezrin (in vitro and in cells) a

Established by other groups independently.

24 25 26 16~

27 28 24 25 30

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Surrey et al. 16 showed that antibodies conjugated with fluorescein isothiocyanate (FITC) are much more efficient for CALI-like inactivation of protein in vitro. We have extended these studies to show that this technique is not only effective in vitro, but in cellular assays as well. 5 We have also shown that the spatial restriction of FALl is sufficient to allow for specific inactivation of proteins in a cellular context. We have named this approach fluorophore-assisted light inactivation (FALl) to distinguish it from CALl. Rubenwolf et al. 17 have used a continuous-wave argon laser for FALl such that each irradiation takes seconds and can be performed in an automated and sequential fashion. Alternatively, Beck et aL 5 have performed FALI with diffuse light such that many samples may be irradiated simultaneously in a 96-well plate. Thus far, several proteins have been inactivated in cells by FALl, including fl 1-integrin, caspase 3, and the Fas receptor.5'17 FALl works by a slightly different mechanism than CALI (Fig. 1B). When FITC is excited, a type 2 photosensitization reaction occurs after excitation with 494-nm light. The transfer of energy from an excited photosensitizer, in this case fluorescein, to an adjacent oxygen molecule generates an excited singlet oxygen molecule. Singlet oxygen is known to have a longer lifetime than free radicals, visualized by extended reactions with neighboring substrates. The upper limit distance for inactivation for FALl has previously been estimated to be less than 300 A.16 We have estimated (using quenching data) that the half-maximal radius of damage is approximately 40 A. 5 Although this value is more than twice as large as the half-maximal radius of damage for CALl, we have shown, using a variety of approaches, that it still shows sufficient specificity such that inactivating neighboring proteins is unlikely. In addition, although it has been shown that singlet oxygen is involved in FALl and that hydroxyl radicals are involved in CALl, there may be other mechanisms involved, as specific quenchers did not completely prevent inactivation. 5'11 FALl provides significant advantages for high-throughput analysis. First, the efficiency of FITC-mediated damage is high enough that a laser is not needed to generate the appropriate light intensity for inactivation. Instead, we can use diffuse light sources such as a desk lamp or a slide projector, such that multiplex irradiation in 96-well plates can be done. In addition, FITC is a significantly less hydrophobic molecule than malachite green isothiocyanate (MGITC). Therefore aggregation during labeling is less problematic and labeling can easily be done in a high-throughput fashion. These advantages allow FALl to be used by a wider range of investigators and can be used to inactivate multiple samples simultaneously. 16 T. Surrey, M. B. Elowitz, E E. Wolf, E Yang, E Nedelec, K. Shokat, and S. Leibler, Proc. Natl. Acad. Sci. U.S.A. 95, 4293 (1998). 17 S. Rubenwolf, J. Niewohner, E. Meyer, C. Corinne Petit-Frere, E Rudert, P. R. Hoffmann, and L. L. Ilag, Proteomics 2, 241 (2002).

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Methods

Antibody Preparation and Labeling with Malachite Green Isothiocyanate and Fluorescein Isothiocyanate We generally employ non-function-blocking antibodies with high affinity for the target of interest for CALI and FALI. Traditional monoclonal or polyclonal antibodies can be used for inactivation, as well as recombinant single-chain variable fragments (scFvs). There is also evidence that ligands for receptors, such as inositol 1,4,5-trisphosphate and malachite green-labeled streptavidin bound to biotinylated enzymes, can also be used for inactivation with high specificity.2,18 In addition, directed inactivation of RNA transcripts using aptamer sequences to bind MG has also been found to be effectiveJ 9 MGITC and FITC are used for labeling and react with amino groups on antibodies to form a stable thioester. Generally, lysine residues in the antibody provide these amino groups. Thus, one must be certain that the antibody preparation does not contain any contaminants with free amino groups, such as Tfis- or glycinebased buffers, because the isothiocyanate group on FITC and MGITC readily reacts with such groups. MGITC is hydrophobic when hydrolyzed, because of its capability for yr-stacking interactions, and thus precipitation may be problematic. This problem is reduced somewhat by keeping the labeling reaction at a high pH (antibody in 0.5 M NaHCO3, pH 9.5), resuspending the MGITC dye in dry dimethyl sulfoxide (DMSO), keeping the MGITC at high concentrations in the labeling mix, and adding bovine serum albumin (BSA) to prevent precipitation. MGITC-labeled BSA has not been found to cause damage to surrounding protein in vitro or in vivo, and thus can be used effectively to reduce hydrophobic MG interactions. A 10-mg/ml solution of MGITC (Molecular Probes, Eugene, OR) in dry DMSO is prepared fresh and added in a 1 : 5 (w/w) ratio with the lgG molecule. We generally label between 100/zg and 1 mg of protein in up to 1 ml of total reaction solution. The MGITC solution is added in three separate aliquots, with constant shaking, every 5 min. After the final addition of MGITC, the labeling mixture is allowed to incubate for 15 min. The free dye is separated from the MG-labeled antibodies by passage through a prepacked gel-filtration column (PD-10; Amersham Pharmacia Biotech, Uppsala, Sweden) in the buffer to be used for the experiment [Hanks' buffered saline (HBSS), phosphate-buffered saline (PBS), Dulbecco's modified Eagle's medium (DMEM), etc.]. We aim to reach a labeling ratio of four to eight dye molecules per lgG or about one or two dye molecules per scFv (a much smaller protein). This value is calculated by taking the optical density of the labeled solution, to determine dye 18 T. Inoue, K. Kikuchi, K. Hirose, M. Iino, and T. Nagano, Chem. Biol. 8, 9 (2001). 19 D. Grate and C. Wilson, Proc. Natl. Acad. Sci. U.S.A. 96, 613l (1999).

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concentration, at 620 nm (molar absorptivity of 150,000 M -1 cm -1) and dividing that value by the antibody concentration. Because of the difficulty in measuring protein concentration after labeling, we assume complete protein recovery (although it is likely less than 100%) and use the optical density (OD) of the solution as a measure of the moles of label. Although this estimate is arbitrary, it is a standard of comparative scale used when determining the labeling versus efficacy for CALl and FALI. For some applications, a concentrated dye solution is required and the MG-labeled antibody solution is concentrated with a Centricon filter (Amicon, Danvers, MA). Storage is generally done in aliquots at - 8 0 °, and is stored for less than 6 months. FITC is less prone to hydrophobic aggregation, and thus labeling is performed in a slightly different manner. Labeling is not often performed in the presence of BSA, because hydrophobic interactions between FITC molecules are less common. To stabilize the FITC-labeled antibody during storage, however, BSA is added to a concentration of 1 mg/ml after labeling. A freshly prepared 10-mg/ml solution of FITC (Molecular Probes) is made in dry DMSO and added to the antibody solution (in 0.5 M NaHCO3, pH 9.5) in a 1 : 5 (w/w) ratio. As with MGITC labeling, we generally label between 100 #g and 1 mg of protein in up to 1 ml of total reaction solution. The FITC solution is added all at once to the antibody solution, and is incubated with constant rocking for 1 hr at room temperature. Free dye is separated from FITC-labeled antibody by gel filtration, using the same protocol as described above for MGITC. A hand-held UV lamp is used to detect fluorescence in the eluate to collect the fractions containing FITC-labeled protein. For FALl, the elution buffer must be phenol red free. Phenol red is a pH indicator added to many culture media. It is an efficient quencher of singlet oxygen species and must be avoided for efficient FALI-generated inactivation. As for MG labeling, the goal is to have four to six FITC molecules per IgG molecule and this can be calculated by dividing the optical density at 494 nm (molar absorptivity of 68,000 M -1 cm -1) by the antibody concentration. The antibody is concentrated with Centricon concentrators, if necessary, and storage is done at - 8 0 ° in aliquots and stored for less than 6 months. FITC labeling of many antibodies in parallel is possible as well. We routinely label 48 scFv molecules at one time in a 96-well plate, and full-chain antibodies could likely be done in the same manner. A solution containing between 50 and 100 #g of antibody in 200/zl is added to a standard 96-well plate along with 2/zl of 10-mg/ml FITC in DMSO and 25/zl of 1 M NaHCO3, pH 9.5. This reaction is incubated for 2 hr at room temperature with rocking. The free dye is removed by passage through G-25 spin columns and associated multiplex-24 plate apparatus (Pharmacia, Piscataway, N J). The labeling ratio is determined by taking the optical density of the FITC-labeled antibody solution at 494 nm in a 96-well Spectrafluor Plus fluorescence plate reader (Tecan, Durham, NC).

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Chromophore-Assisted Laser Inactivation: Laser Setup and Irradiation Conditions

Nd:YAG (neodymium-doped yttrium aluminum garnet)-driven dye laser (GCR-11 with HG-2 doubling crystal, PDL-2; Spectra-Physics, Mountain View, CA) DCM laser dye in methanol (Exciton, Dayton, OH): oscillator concentration, 175 rag/liter; amplifier concentration, 40 mg/liter Right-angle prism, holder, and mounting rods (Newport, Fountain Valley, CA) Convex lens and lens holder (Newport) Laser parameters are as follows: peak power, 56 MW/cm2; spot size, 2 mm; pulse width, 3 ns; frequency, 10 Hz; energy per pulse, 15 mJ. An Nd:YAG-driven dye laser (GCR-11 with HG-2 doubling crystal, PDL-2; Spectra-Physics) along with DCM laser dye in methanol (Exciton; oscillator dye, 175 mg/liter; amplifier dye, 40 mgfliter) is used to generate the pulsed 620-nm laser light. The laser is mounted on a vibration-free table (Newport) and the laser beam is directed from the Nd:YAG-pumped dye laser to a right-angle prism mounted on a rod and prism holder approximately 50 cm from the exit port. The laser beam is directed down vertically onto the center of an interjected planar-convex lens (focal length, ~70 mm) attached to the same mounting rod as the prism. The laser spot size is determined by the distance between the prism and the lens, and is usually set to a size of 2 mm. The spot size can be determined by placing a black-and-white Polaroid photographic print on the table below the lens. Each pulse (3-ns pulse width) of the laser generates approximately 15 mJ of energy and forms a single, slightly oblong, uniformly bleached spot of 2 mm. Protein or suspended cell samples to be used for CALl in vitro are placed in the wells of a Nunc transferrable solid-phase plate (Nunc International, Roskilde, Denmark). The wells of the plate are approximately 2 mm in diameter, and the laser beam should be centered as accurately as possible in the well. Pulsed irradiation is allowed to occur for 2 to 5 min, depending on the assay to be done. In vitro experiments are usually pulsed for 5 min, whereas assays involving cells are usually limited to 2 min. For cell-based assays and in vitro assays of temperaturesensitive enzymes, samples are incubated on ice during irradiation to reduce sample heating. Whenever lasers are used, extreme caution is employed, with special concern for eye protection. The short pulse width of the Nd:YAG laser produces high peak power (megawatts) and a single stray beam could cause blindness. Protective goggles are always worn, unless specified, and beam blockers are placed to prevent stray reflections. Contact with skin should also be avoided. Investigators are advised to carefully follow laser safety protocols provided with the laser system.

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Fluorophore-Assisted Light Inactivation: Slide Projector Setup and Irradiation Conditions Ektographic III slide projector (Kodak, Rochester, NY) Brilliant blue filter 69 (Roscolux, Stamford, CT) Rectangular mirror, holder, and mounting rods (Newport) Light parameters are as follows: power, 300 W. Because FITC absorbs visible light (494 nm) and is more efficient than MGITC for inactivation, it is possible to use a variety of light sources for FALl. We have shown that continuous-wave laser light (argon ion) or diffuse light from an ordinary 60-W light bulb or from a 300-W slide projector are all effective light sources for FALl. 5 Routinely, we use a 300-W slide projector containing a blue filter in the slide slot, which limits the transmission of light so that greater than 50% of the transmitted light is between 420 and 500 nm (Brilliant blue filter 69; Roscolux). Our setup is illustrated in Fig. 2. The slide projector is set up such that the light is directed onto a mirror that is oriented at a 45 ° angle approximately 19 cm from the projector and 10 cm from the sample. The light is thus projected downward onto the sample. Many samples can be illuminated at one time if a multiwell plate is used, because the projected light is not focused into a beam. However, the

19cm

FIG. 2. The setup for FALI using a diffuse light source. A standard 300-W slide projector is raised approximately 10 cm from a base and focused onto a mirror attached to a stationary stand at an ~45 ° angle from the base. A blue filter (Brilliant Blue 69; Roscolux, Stamford, CT) is placed into the slide slot to restrict the transmission of light so that >50% of light is between 420 and 500 nm. The middle of the mirror is placed about 20 cm from the lens of the projector. The sample to be illuminated is placed under the mirror on ice, measured about 15 cm from the top of the mirror. Illumination is allowed to occur for 1 hr for cellular assays or for 30 rain for in vitro assays.

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irradiation time is much longer when using a diffuse light source than when using focused laser light. The q/2 for inactivation is --~10 min. 5 It is useful to perform a dose-response curve for FALl. To achieve maximal inactivation in cellular assays, samples are illuminated for 1 hr while incubated on ice to reduce sample heating. Generally, in vitro FALI assays are illuminated for 30 min for maximal inactivation.

Applying Fluorophore-Assisted Light Inactivation in Multiplex Assay for Cancer Cell Invasion The multiplex capacity of FALI allows us to assess in parallel the role of many proteins in cellular processes using an array of antibody or scFv antibodies. We have coupled FALI to a cancer cell invasion assay for this purpose and tested it with antibodies against ~l-integrin, a well-validated protein target involved in invasiveness. 5'2° Cancer cell invasiveness is an area of high clinical importance as an early step in metastasis. A standard assay for cancer invasiveness is the in vitro transwell assay. 21 The transwell assay is based on the ability of cells to migrate through an 8-/~m pore size filter coated with Matrigel, an active fraction of extracellular matrix that mimicks the basement membrane in cell culture. 22 Cells that migrate through the Matrigel are manually counted or measured colorimetrically after manual removal of the cells on the top layer (i.e., cells that fail to migrate). 23 The transwell assay discriminates well between invasive and noninvasive cells and there is an excellent correlation between cells that can cross the Matrigel-coated filter and metastasize in nude mice. 21 This assay has also been used to test the efficacy of anti-invasiveness drugs such as doxorubicin. Our assay incorporates several existing technologies to generate a highthroughput version of the transwell assay compatible with FALI. Instead of the transwell, we employ a plate-sized filter (Neuroprobe, Gaithersburg, MD) with a 96-holed mask chamber. We use CellTracker Orange labeling (Molecular Probes) of cells (fluorescently labeled, but at a different absorption maximum than FITC) to quantitate the number of cells that cross the filter. After the invading cells cross the filter, the top side of the filter is scraped in single vertical and horizontal sweeps that are amenable to automation. The fluorescence of the underside of the filter is then quantitated in parallel using a fluorescence plate reader. This assay has several advantages over the conventional transwell assay. It is significantly less expensive per sample (approximately one-tenth the cost per single assay) and utilizes a fluorescent plate reader to read many samples on a filter simultaneously so 2o R. Fassler, M. Pfaff, J. Murphy, A. A. Noegel, S. Johansson, R. Timpl, and R. Albrecht, J. Cell Biol. 128, 979 (1995). eL A. Albini, Y. Iwamoto, H. K. Kleinman, G. R. Martin, S. A. Aaronson, J. M. Kozlowski, and R. N. McEwan, Cancer Res. 47~ 3239 (1987). 22 V. P. Zerranova, E. S. Hujanen, D. M. Loeb, G. R. Martin, L. Thornburg, and V. Glushko, Proc. Natl. Acad. Sci. U.S.A. 83, 465 (1986). 23 K. Saito, T. Oku, N. Ata, H. Miyashiro, M. Hattori, and I. Saiki, Biol. Pharm. Bull. 20, 345 (1997).

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that the results of an entire plate of assays can be read simultaneously. In addition, the volume is significantly lower than currently used for transwell assays, which allows the use of the assay for samples that are cell limited (e.g., clinically derived tumor samples). We currently assess 40,000 cells per assay and have evidence that fewer cells can be used with equal sensitivity.

Fluorophore-Assisted Light Inactivation: Coupled to Transwell Assay for Invasion CellTracker Orange (Molecular Probes) ChemoTx disposable chemotaxis system (Neuroprobe) Matrigel (Becton Dickinson, Franklin Lakes, NJ) Spectrafluor Plus fluorescence plate reader (Tecan) Preparation of the membrane for assaying invasion is a critical step. Matrigel (13.3 #1 of a 0.300-mg/ml solution in PBS) is coated on top of the membrane. The membrane is then placed in a desiccator (humidity, <18%) overnight to dry. At least 2 hr before initiation of the assay, the Matrigel-coated wells are rehydrated with 0.1% (w/v) BSA-DMEM. Also before the assay, the cells are fluorescently labeled with CellTracker Orange (Molecular Probes). This process is done essentially as described by the manufacturer, with minor modifications. First, the cells are washed with 0.1% (w/v) BSA-DMEM. After the wash, a 3#M solution of CellTracker Orange (Molecular Probes) is added to the flask and incubated for 15 rain. After this incubation, the cells are washed twice with 0.1% (w/v) BSA-DMEM and then allowed to recover in 0.1% (w/v) BSA-DMEM for an additional 15 min. To remove the cells from the flask, they are washed with HBSS (no Ca 2+ or Mg 2+) once and then with 0.53 mM EDTA once. A small amount of 0.53 mM EDTA is then added back to the cells and allowed to incubate for 6 min at 37 ° and 7% CO2. The cells are removed by gently washing the flask with 0.1% (w/v) BSADMEM. The cell suspension is subsequently pelleted at 1000 rpm, washed with 0.1% (w/v) BSA-HBSS (no phenol red), and repelleted. The pellet is resuspended in 0.1% (w/v) BSA-HBSS (no phenol red) and the cells are counted by trypan blue exclusion for viability. The cells are brought to a concentration of 8 × 106 cells/ml and an equal volume of 40-/zg/ml antibody solution [in 0.1% (w/v) BSA-HBSS (no phenol red)] is added. The cells are aliquoted in 38-/zl volumes to two nontissue culture-treated 96-well plates and allowed to incubate for 1 hr. The plates are subsequently put on ice, and one plate is illuminated by Brilliant Blue filtered slide projector light for 1 hr. After illumination, 0.1% (w/v) BSA-DMEM is added to each well to a cell concentration of 8 x 105. Chemoattractant [5% (v/v) FCS in DMEM] is added to the bottom plate and the membrane is placed on top after removing hydration medium. Fifty microliters (40,000 cells) of the cell-antibody mixture is added to each well of the membrane and the membrane is incubated for 6 hr at 37 ° and 7% CO2. After incubation, the top of the membrane is scraped with

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a cell lifter, rinsed with HBSS, swabbed with a cotton-tipped applicator, and dried with Whatman (Clifton, NJ) paper. The membrane is then read with a fluorescence plate reader. 23 Concluding Remarks FALI allows us to apply light-mediated protein inactivation to samples in a multiplex fashion to address protein function in cellular processes with potentially high throughput. Each step is compatible with robotic automation, and this should increase throughput and decrease variance. Although FALl is an effective technique for protein inactivation, many aspects of development of FALI are still in progress. For example, although the FITC dye is effective for FALI, it has low quantum efficiency for singlet oxygen generation (--,2%). As such, more efficient light-induced singlet oxygen generators may allow for shorter irradiation times, more potent inactivation, and higher throughput. We expect that innovations in the selection of dyes and the possible use of expressed fluorophores (such as green fluorescent protein or its variants) will extend the variety of applications for highthroughput FALl. These types of tools will prove to be necessary when addressing the complex functional roles of the proteome. Acknowledgments The authors acknowledge support from NIH Grants CA81668, NEI 11992, and NS34699, a predoctoral training grant DK07542 to B.K.E., and the GRASP Center at the New England Medical Center for monoclonal library preparation. FALl was codeveloped and tested with Xerion Pharmaceuticals (Martinsried, Germany). 24 E S. Wang, J. S. Wolenski, R. E. Cheney, M. S. Mooseker, and D. G. Jay, Science 273, 660 (1996). 25 A. M. Sydor, A. L. Su, F. S. Wang. A. Xu, and D. G. Jay, J. Cell Biol. 134, 1197 (1996). 26 L. Castelo and D. G. Jay, Mol. Biol. Cell 10, 1511 (1999). 27 R. E Lamb, C. Roy, T. J. Diefenbach, H. V. Vinters, M. W. Johnson, D. G. Jay, and A. Hall, Nat. Cell Biol. 2, 281 (2000). 28 C. W. Liu, G. Lee, and D. G. Jay, Cell Motil. Cytoskel. 43, 232 (1999). 29 K. Takei, R. M. Shin, T. Inoue, K. Kato, and K. Mikoshiba, Science 282, 1705 (1998). 30 R. F. Lamb, B. W. Ozanne, C. Roy, L. McGarry, C. Stipp, P. Mangeat, and D. G. Jay, Curr. Biol. 7, 682 (1997). 31 D. G. Jay and H. Keshishian, Nature (London) 348, 548 (1990). 32 D. Schmucker, A. L. Su, A. Beermann, H. Jackle, and D. G. Jay, Proc. Natl. Acad. Sci. U.S.A. 91, 2664 (1994). 33 K. Takei, T. A. Chan, E S. Wang, H. Deng, U. Rutishauser, and D. G. Jay, J. Neurosci. 19, 9469 (1999). 34 j. j. Feigenbaum, M. D. Choubal, K. Payza, J. R. Kanofsky, and D. S. Crumrine, Peptides 17, 991 (1996). 35 M. Schroeder, S. Miller, V. Srivastava, E. Merriam-Crouch, S. Holt, V. Wilson, and D. Busbee, Mutat. Res. 316, 237 (1996). 36 R. Schroder, D. G. Jay, and D. Tautz, Mech. Dev. 80, 191 (1999).