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Flow Cytometric Analysis of Myometrial and Decidual Cell Suspensions
Flow Cytometric Analysis of Myometrial and Decidual Cell Suspensions
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Adam Blaisdell Department of Pathology, NYU School of Medicine, NY, USA
Adrian Erlebacher Department of Pathology, NYU School of Medicine, NY, USA; NYU Cancer Institute, NYU School of Medicine, NY, USA CHAPTER SUMMARY Flow cytometry is a powerful tool with single-cell resolution that allows the researcher to gain information on the cellular composition of a tissue and to analyze multiple cell subsets at once. Here, we provide a methodological framework for applying flow cytometry to the study of leukocyte biology in the pregnant mouse uterus.
Abbreviations
PBS
phosphate buffered saline
7-AAD
7-aminoactinomycin D
PE phycoerythrin
ACK
ammonium chloride potassium
PES polyethersulfone
APC allophycocyanin
RBC
red blood cell (erythrocyte)
BrdU bromodeoxyuridine
RT
room temperature
SSC
side scatter
uNK
uterine natural killer
BSA
bovine serum albumin
Cy7
cyanine 7
ddH2O
double-distilled water
DBA
Dolichos biflorus lectin
DC
dendritic cell
FACS
fluorescence-activated cell sorting
FBS
fetal bovine serum
FITC
fluorescein isothiocyanate
FSC
forward scatter
HBSS
hank’s balanced salt solution
MHCII
class II major histocompatibility complex, which presents peptide antigens to CD4 T cells
Overview Many hematopoietic and nonhematopoietic cell types reside within peripheral tissues, and the pregnant mouse uterus is no exception. Multicolor flow cytometry offers a powerful tool to simultaneously analyze all of these cell populations using combinations of fluorescently labeled antibodies directed against different surface antigens. This technique is the analytic version of fluorescence-activated cell sorting (FACS), which is used to purify live cells. In this chapter, we provide a methodological framework for applying flow cytometry to the study of uterine leukocytes, with emphasis on approaches for dividing the cells
The Guide to Investigation of Mouse Pregnancy. http://dx.doi.org/10.1016/B978-0-12-394445-0.00053-9 © 2014 Elsevier Inc. All rights reserved.
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(A) SSC
7-AAD
Viable leukocytes
FSC
MHCII+ monocytes
CD45
MHCII
CD11c
CD11blo DCs CD11bhi DCs
MHCII– monocytes
CD11b NK cells
ev I iv MHCIIhi macrophages MHCIIlo macrophages
MHCII
F4/80
Ly6C
FSC
MHCII
MHCII
Gr-1
SSC
Neutrophils
ev I iv
F4/80
FSC
CD11b
SSC
T cells
SSC
SSC
CD11c
Eosinophils
FSC
FSC
(B) SSC
7-AAD
Viable leukocytes
FSC
MHCIIhi monocytes
CD45
MHCII
CD11c
CD11blo DCs CD11bhi DCs
F4/80
FSC
T cells CD11b
ev I iv MHCIIhi macrophages MHCIIlo macrophages ev I iv
SSC
SSC
CD11c
CD11b NK cells
MHCIIlo monocytes
MHCII
F4/80
Ly6C
FSC
MHCII
MHCII
Gr-1
SSC
Neutrophils
FSC
Figure 1 Flow cytometric identification of leukocytes in gd 9.5 implantation sites of pregnant C57BL/6 mice. The myometrium (A) was dissected away from the decidua (B) prior to tissue digestion and analysis by a 3-laser, 10-color BD LSRII flow cytometry. The figure is meant to demonstrate a general gating scheme that allows one to identify as many different important leukocytes as possible, with emphasis on myeloid cells. Alternate gating schemes, additional criteria using the markers shown, and the use of additional markers (as permitted by the number of available fluorescence channels) will allow for greater specificity. For example, it is not appropriate to rely upon this gating strategy to identify T cells and NK cells because these cells should be identified using antibodies to CD3, TCRβ, and CD122. The gating scheme shown employed the following antibody-fluorochrome conjugates: CD45-Pacific orange; Ly6C-APC; Gr-1-Alexa 700; F4/80-FITC; MHCII-Pacific blue; CD11b-PE; CD11c-PE-Cy7. The PE-Texas Red and APC-Cy7 channels were not used. CD45-Pacific Orange and Gr-1-Alexa 700 are preferred conjugates because their corresponding fluorescence channels (AmCyan and Alexa 700, respectively) are dim. Summary of the gating strategy: After a broad gating on FSC vs SSC to include all possible leukocytes, viable leukocytes are identified as CD45+ 7-AAD− cells. Neutrophils are then identified as Gr-1hi Ly6Cint cells, which greatly simplifies the subsequent specific visualization of Ly6Chi monocytes. Because of the dimness of Alexa 700 fluorochrome, the neutrophils
FLOW CYTOMETRY PROTOCOL CHAPTER 53
into their major subsets. We will not provide a comprehensive overview of flow cytometry because this can be found elsewhere in many books (e.g., Current Protocols in Immunology). Flow cytometry can also be used to determine expression levels of intracellular proteins, DNA content, and proliferation status. However, these techniques are also outside the scope of this chapter and are discussed in detail elsewhere. The importance of flow cytometry in studying uterine leukocyte biology during pregnancy is becoming increasingly evident. Most strikingly, the technique allows for the direct visualization, and thus the discovery, of uterine leukocyte subsets. For example, we recently used this technique to show that uterine macrophages divide into two subsets based upon their surface expression levels of class II major histocompatibility complex, which presents peptide antigens to CD4 T cells (MHCII).1 These two subsets show differential expression of several genes involved in tissue remodeling, thus suggesting they may have unique nonredundant roles in the pregnant uterus.1,2 Flow cytometry has also uncovered two subsets of uterine natural killer (uNK) cells within the pregnant mouse uterus that share expression of the interleukin-2 receptor β subunit (CD122) but differ in their binding to Dolichos biflorus agglutinin (DBA).3 As with the aforementioned macrophages, these uNK cells show distinct expression profiles and may therefore execute different functions during pregnancy. When combined with measurements of tissue weight, flow cytometry can also be used to calculate both leukocyte tissue densities within uterine tissue layers as well as absolute leukocyte number per implantation site. Density, rather than absolute number, likely has the greater functional relevance. The importance of this distinction can best be exemplified by the correlation of certain leukocyte densities
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with prognosis in growing tumors4 and likely holds true for rapidly growing tissues, such as the myometrium and decidua. Indeed, macrophage densities in the myometrium remain relatively constant throughout pregnancy, despite the fact that their absolute numbers per implantation site increase drastically; conversely, decidual macrophage densities drop considerably without a significant change in absolute number per implantation site.1 It is therefore likely that the decidua has mechanisms in place to control macrophage densities (as opposed to absolute number), possibly in an effort to minimize the occurrence of an adverse inflammatory reaction that could compromise the health of the fetus. Flow cytometry can also be used to discriminate intravascular vs extravascular leukocytes (Section Determination of Uterine Leukocyte Blood/Tissue Partitioning).1 This turns out to be an important distinction in the context of the pregnant mouse uterus because several leukocyte subsets (e.g., monocytes) show considerably lower rates of extravasation into the decidua as compared to the myometrium under steady-state conditions. As a result, the decidua contains a large number of monocytes within its intravascular compartment, whereas myometrial monocytes are mainly extravascular (see Figure 1). The technique we describe here to reveal leukocyte blood/tissue partitioning involves intravenous injection of a low dose of pan-leukocyte (anti-CD45) antibodies several minutes prior to sacrifice, thus allowing for the select labeling of all intravascular cells. This technique provides a distinct advantage over vascular perfusion because some decidual leukocytes, including monocytes, remain adherent to the decidual vasculature even after perfusion.1,5,6 The flow cytometric analysis of peripheral tissue leukocytes requires the preparation of single-cell
here appear simply as Gr-1+, while Ly6Chi monocytes appear Gr-1− even though they express this marker at fairly high levels. Ly6Chi monocytes divide into MHCII− and MHCII+ subsets. In the myometrium, Ly6Chi monocytes are predominantly extravascular. This is particularly the case for MHCII+Ly6Chi monocytes, which also show some F4/80 upregulation presumably reflecting an initial step in their differentiation into macrophages. In contrast, Ly6Chi monocytes in the decidua are predominantly intravascular, and appear to have undertaken a differentiation program of MHCII and F4/80 upregulation even within the intravascular compartment. Macrophages, which are Ly6Cneg-intF4/80+ cells and divide into MHCIIlo and MHCIIhi subsets, are extravascular in both the myometrium and decidua. DCs are identified as Ly6Cneg-intMHCII+F4/80−CD11chi cells that divide into CD11blo (CD103+; not shown) and CD11bhi (CD103−; not shown) subsets. We prefer a stringent CD11chi gate for CD11bhi DCs to exclude as many macrophages as possible because the prior MHCII vs F4/80 gate used to collectively identify DCs will not exclude all macrophages. Eosinophils are F4/80lo cells that show a characteristic high SSC profile; note that these cells are absent from the decidua. After excluding all the myeloid populations listed above, uNK cells are within the remaining CD11b+ CD11c+ population; T cells are within the CD11b−CD11c− population. Note that uNK cells are more granular than T cells. MHCII+F4/80−CD11b−CD11c− cells in the decidua are probably B cells. In a separate experiment, CD45-PE-Cy7 antibodies (1 μg) were intravenously injected 5 min before sacrifice in order to discriminate intravascular (iv) from extravascular (ev) macrophages and monocytes. These plots used a different set of antibody-fluorochrome conjugates for cell identification (not shown).
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suspensions. This typically involves a step of limited proteolysis to disrupt cell–cell interactions and to release leukocytes from their attachments to the extracellular matrix. This treatment, however, must maintain the integrity of all surface antigens of interest. To this end, Liberase TM Research Grade (Roche) has proven most effective in our hands. Within 30 min, both myometrium and decidua can be reduced to a virtual single-cell suspension; to our knowledge, no relevant epitopes have been compromised. If the analysis requires a complete analysis of nonleukocytes as well, trypsin can be added to the Liberase cocktail to allow complete release of stromal and endothelial cells. Trypsin, however, destroys many surface epitopes, thus limiting the discrimination of many cell types and in-depth analysis of surface phenotypes. However, we have found that trypsin does not destroy the epitopes recognized by antibodies directed against CD45, ICAM-2 (CD102), and EpCAM (CD326), thus allowing the broad discrimination of leukocytes (CD45+), endothelial cells (CD45− CD102+ CD326−), epithelial cells (CD45− CD102+ CD326+), and stromal cells (CD45− CD102− CD326−).7 Following data collection, the typical first step of a flow cytometric analysis is to display the cells on a side scatter (SSC; a measure of cell granularity) vs forward scatter (FSC; a measure of cell size) plot (Figure 1). This allows the researcher not only to gate away cell debris and red blood cells (RBCs), but also to enrich for leukocytes because they tend to be smaller than
nonleukocytes. However, we have found it to be extremely useful to include the panleukocyte marker CD45 in our staining cocktail in order to definitively identify leukocytes and avoid the analytic complications that otherwise arise when using certain markers (e.g., Ly6C) that are expressed by both leukocytes and nonleukocytes. We typically use an anti-CD45 Pacific Orange conjugate (Table 1) because the corresponding fluorescent channel (AmCyan) is quite dim and thus unusable for many other markers. We also use a live-dead discrimination reagent (e.g., propidium iodide or, preferably, 7-aminoactinomycin D (7-AAD)) in order to gate away dead cells, which can be autofluorescent and thus appear to express many surface markers. Finally, if density analyses are performed, it is important to maintain consistency in cell preparation and total live cell determination in order to be able to combine results from multiple experimental runs. We determine the total number of live cells in our suspensions using a hemocytometer and trypan blue exclusion. Finally, one fluorescent channel (e.g., PE-Texas Red, which is otherwise not useful) can be left open to allow for identification of naturally autofluorescent cells. In the late-gestation pregnant mouse uterus, autofluorescent cells appear as a wisp emanating from the CD45+ population. These cells are primarily of fetal origin and should be gated away, thereby preserving an exclusive analysis of maternal leukocytes. Given the above basic principles, more specific analytic strategies can be developed, depending
Table 1 Example of Staining Panel Used to Identify Myeloid Cells in Myometrial and Decidual Tissue as well as to Discriminate Intravascular vs Extravascular Leukocytes Fluorochromea
Epitope
Supplier
Clone
Catalog Number
Dilution
Fluorescein isothiocyanate
Ly6C
Biolegend
HK1.4
128005
1:400
Phycoerythrin (PE)
F4/80
Biolegend
BM8
123109
1:100
PE-Texas Redb
–
–
–
–
–
7-AAD (PerCP-Cy5.5)
–
BD Pharmingen
–
51-68981E
1:100
PE-Cy7
CD45 (intravascular)
Biolegend
30-F11
103113
–
Pacific blue
MHCII (I-A/I-E)
Biolegend
M5/114.15.2
107619
1:400
Pacific orange (AmCyan)
CD45 (total)
Invitrogen
30-F11
MCD4530
1:100
Allophycocyanin (APC)
CD11c
Biolegend
N418
117309
1:200
Alexa 700
Gr-1c
Biolegend
RB6-8C5
108421
1:200
APC-Cy7
CD11b
Biolegend
M1/70
101225
1:200
aBased
upon fluorescent channels available on 3-laser (violet: 405 nm; blue: 488 nm; red: 633 nm) 10-color LSRII flow cytometer (BD Bioscience). fluorescent channel left open to identify autofluorescent cells. cCan be replaced with anti-Ly6G antibodies, clone 1A8. bThis
FLOW CYTOMETRY PROTOCOL CHAPTER 53
upon whether the user wishes to analyze many cell subsets at once or perform an in-depth surface phenotype analysis of one specific subset. The most sophisticated flow cytometry currently available can track up to 18 different markers. For most analyses, a 10-color format based on the use of a three-laser flow cytometer—violet (405 nm), blue (488 nm), and red (633 nm)—suffices for identification of all cell subsets within the mouse uterus, and the protocols and schema described herein are based upon this format. Indeed, using 10-color flow cytometry, virtually all cells of myeloid origin (macrophages, dendritic cells (DCs), monocytes, neutrophils, eosinophils) can be identified in one staining scheme (Table 1, Figure 1), and a similar approach can be used for cells of lymphoid origin. However, any single population can typically be identified with fewer than 10 markers, which thus allows for further phenotypic characterization using the open fluorescent channels.
Protocol Equipment, Mice, and Supplies 1. Equipment a. Flow cytometer, LSRII (BD Biosciences), three-lasers (405, 488, 633 nm) b. Cell acquisition software (BD Biosciences FACSDiva) c. FACS data analysis software, such as FlowJo (Tree Star) or FCS Express (De Novo Software) d. Microcentrifuge, refrigerated e. Water bath, 37°C 2. Dissection microscope and light source 3. Instruments a. Dissection/mincing tools Scissors, straight, 14.5 cm (Fine Science Tools #14000-14) Scissors, extra narrow, 10.5 cm (Fine Science Tools #14088-10) Scissors, fine, curved, 10.5 cm (Fine Science Tools #14061-10) Graefe forceps, straight, 10 cm (Fine Science Tools #11050-10) Dumont #5 forceps, straight, 11 cm (Fine Science Tools #11251-20) b. Pipetters (1 ml, 100 μl, 10 μl) with tips c. 10 cm Petri dish (BD Falcon #351029) d. 1.7 ml microcentrifuge tubes (Denville Scientific #C2170)
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e. Sterile disposable filter, polyethersulfone (PES) membrane (90 mm), 0.2 μm pore size (Thermo Scientific #569-0020) f. Cell strainer, nylon mesh, 70 μm (BD Falcon #352350) g. Hematocytometer and light microscope h. 5 ml FACS tube, polystyrene, round bottom (BD Falcon #352052) i. (Optional) Insulin syringe (Becton Dickinson #309301) 4. Nonpregnant or pregnant mice, or mice with artificial deciduas 5. Reagents a. Hanks’ Balanced Salt Solution (HBSS) with Ca2+ and Mg2+ (Invitrogen #14025-076) b. Liberase TM Research Grade (Roche #05401127001), diluted to 28 Wunsch units (WU)/ml (100×) in double-distilled water (ddH2O) and stored in 50 or 100 μl aliquots at −20°C; thawed as needed and used immediately c. DNase I (Roche #10104159001), diluted to 30 mg/ml (1000×) in ddH2O and stored in 5–10 μl aliquots at −20°C; thawed as needed and used immediately d. (Optional) Trypsin, 2.5%, no phenol red (Invitrogen #15090-046, 50×) stored in 100 or 200 μl aliquots at −20°C; thawed as needed and used immediately e. (Optional) Ammonium chloride potassium (ACK) lysing buffer (Lonza #10-548E) f. Phosphate-buffered saline (PBS) without Ca2+ and Mg2+ (Cellgro #21-031-CV) g. Bovine serum albumin (BSA; Sigma #A2153) h. Trypan Blue (Thermo Scientific #SV30084.01) i. FcγR blocking antibody (anti-CD16/32; Clone 2.4G2). This reagent is available from multiple vendors. We buy ours in bulk from BioXCell (#BE0008). The antibody is aliquoted at 1 mg/tube and stored at −80°C until needed, then diluted to 1 mg/ml in PBS and stored at 4°C j. (Optional) RPMI 1640 medium (Invitrogen #21870-076) k. (Optional) Fetal bovine serum (FBS; Atlanta Biologicals #S12450), heat inactivated for 30 min at 55°C l. (Optional) Heparin sodium, 1000 USP units/ ml (Sagent Pharmaceuticals #400-01) 6. Solutions
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a. Tissue digestion buffer (example of 10 ml preparation) 9.89 ml HBSS containing Ca2+ and Mg2+ 100 μl Liberase TM (28 WU/ml); final concentration 0.28 WU/ml 10 μl DNase I (30 mg/ml); final concentration 30 μg/ml (Optional) 200 μl Trypsin (2.5%); final w:v 0.05%; subtract from HBSS volume accordingly b. FACS buffer (example of 500 ml preparation) 495 ml PBS without Ca2+ and Mg2+ 5 g BSA; final w:v 1% 5 ml 0.5 M EDTA, pH 8.0; final concentration 5 mM Filter buffer through PES membrane with 0.2 μm pore size prior to use in order to sterilize and degas. c. (Optional) RPMI-10 (example of 500 ml preparation) 450 ml RPMI 1640 medium 50 ml FBS Filter buffer through PES membrane with 0.2 μm pore size prior to use. d. (Optional) Intravascular leukocyte labeling solution (100 μl per mouse) 90 μl PBS without Ca2+ and Mg2+ 5 μl Heparin sodium, 5 USP units total 5 μl anti-mouse CD45 (Clone 30-F11, 0.2 mg/ml), 1 μg total, preferably conjugated to a brighter fluorochrome such as PE (Biolegend #103105), PE-Cy7 (Biolegend #103113), or allophycocyanin (APC; Biolegend #103111)
Methods General Leukocyte Analysis
1. Tissue digestion buffer is freshly prepared (see Section Equipment, Mice, and Supplies) and stored on ice. 2. Mice are euthanized according to the institutionally approved protocol. Uteri are immediately dissected: fine straight dissection scissors are used to sever the cervical attachment, after which the cervix is held with Graefe forceps in a raised position, allowing the mesometrium to be peeled away with Dumont no. 5 forceps; both fallopian tube attachments are then severed to completely release the uterus, which is then immersed in cold HBSS within a 10 cm Petri dish.
3. Using the dissection microscope and light source, the mesometrium is trimmed off the uterus, and the horns are divided at the cervix. Individual implantation sites are then separated from each other by cutting at the point where the interimplantation site directly abuts the decidua; the interimplantation sites are then d iscarded unless they are to be analyzed in parallel with the myometrium and decidua. For gestation day (gd) 4.5 and gd 5.5, the myometrium and decidua cannot be separated, and these tissues are thus analyzed together (see Chapter 1). By gd 6.5, however, the myometrium can easily be separated from underlying decidua, as these tissues are only tightly attached at the mesometrial pole. The free edge of the myometrium is grasped with Dumont no. 5 forceps at the point where the interimplantation site was severed, and snipped once with fine scissors to increase the size of the hole (see Chapter 2). The myometrium is then peeled away from the decidua by threading it through Graefe forceps. The embryo and yolk sac are removed from the interior of the decidua using Dumont no. 5 forceps. This procedure is performed for each implantation site separately. At gd 11.5 and later, the placenta will comprise a significant fraction of the dissected “decidual” tissue. Although it is possible to separate decidua from placenta, this process is difficult, time-consuming, and imprecise. Therefore, after discarding the fetus, its amniotic and yolk sac membranes and the umbilical cord, the decidua, and placenta are taken together for further analysis. The decidual localization of a cell type of interest, however, needs to be confirmed by tissue immunostaining. 4. The myometrium and decidua (or combined decidual/placental tissue) from each litter are separately pooled into 1 ml tissue digestion buffer within a microcentrifuge tube at no more than 100 mg tissue/ml. If the amount of t issue exceeds 100 mg, multiple microcentrifuge tubes are prepared to accommodate the excess. Using an insufficient amount of tissue digestion buffer will lead to incomplete digestion, which could compromise the results of the e xperiment. If necessary to study rare cell populations, specimens from multiple litters can also be pooled. 5. Tissues are minced within the microcentrifuge tube with fine curved dissection scissors until large chunks are no longer visible.
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6. Samples are incubated in a water bath at 37°C for 45 min. After the first 15 min, and then every 5–10 min thereafter, the tissue fragments are disaggregated by pipetting the solution m ultiple times through a 1 ml pipet tip. Ultimately the solution should resemble a soup and should be easily pipetted. If not, the tubes can be incubated for an additional 15 min at 37°C. Special consideration: If trypsin is additionally added to the tissue digestion buffer in order to generate single-cell suspensions of stromal cells and endothelial cells, the incubation time should be increased to 60 min. EDTA (to 5 mM final concentration) should be added for the last 15 min in order to inhibit Ca2+- and Mg2+-dependent adhesive interactions, with the solution again pipetted every 5–10 min. 7. Samples are centrifuged at 1000×g for 4 min at 4°C to pellet cells. Supernatant is aspirated. 8. (Optional; recommended for decidua) Although abundant, RBCs are not an interesting cell population within the uterus and can therefore be lysed in an effort to “purify” all other cells and thus speed data acquisition on the flow cytometer. RBC lysis is especially important for the blood-rich decidua. Samples are r esuspended in 1 ml ACK lysing buffer and promptly transferred to a 15-ml conical tube. Then, 9 ml of ice-cold RPMI-10 is immediately added to stop the lysing reaction. Samples are centrifuged at 1000×g for 4 min at 4°C to pellet cells. Supernatant is aspirated, and the resuspended pellet is washed again with 10 ml RPMI-10. If it is observed that a significant number of RBCs remain after lysis, the lysing reaction can be continued for a longer time before the addition of RPMI-10. However, the lysing reaction can potentially destroy leukocytes and nonleukocytes. Thus, it should be performed on ice and not allowed to continue for longer than 5 min. 9. Samples are resuspended in 1 ml FACS buffer and incubated in a water bath at 37°C for 15 min, and then pipetted several times though a 1-ml pipet tip. This step serves to further disaggregate the cells by inhibiting Ca2+- and Mg2+dependent adhesive interactions. 10. Samples are filtered through a 70-μm nylon mesh cell strainer into a new microcentrifuge tube. 11. Samples are centrifuged at 800×g for 4 min at 4°C to pellet cells. Supernatant is aspirated. 12. Samples are resuspended in 1 ml FACS Buffer and 10 μl is taken for a cell count. Samples are
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centrifuged at 800×g for 4 min at 4°C to pellet cells. Supernatant is aspirated. Although cell counts are essential for cell density analysis, they should be performed for all experimental formats to ensure optimal labeling of cells with antibodies (Steps 13, 16). 13. Samples are resuspended in Fc blocking buffer (see below) at a concentration no greater than 106 cells per 100 μl buffer and incubated on ice for 10 min. Even if there are fewer than 106 cells in a given sample, a minimum of 100 μl buffer should be used. For both the Fc blocking and labeling steps (Steps 16, 19), it is important to keep a relatively low cell density in order to sufficiently block and label all cells. If the n umber of cells in a given sample greatly exceeds 106, one of two options can be chosen: (1) A smaller aliquot of the sample (<106 cells) is taken and used to represent the entire sample. This option is useful when analyzing cell subsets that are relatively abundant within the tissue; therefore, only a small number of cells will need to be acquired by the flow cytometer. (2) A larger amount of Fc blocking buffer (and staining b uffer in Step 16) can be added to the entire sample to maintain a relatively low cell density. This option is useful when analyzing rare cell subsets that require acquisition of a large number of total cells. Example of 1 ml preparation of Fc blocking buffer: 990 μl FACS buffer 10 μl FcγR blocking antibody (1 mg/ml); final concentration 10 μg/ml 14. Samples are brought to 1 ml with FACS buffer. Small amounts of each sample are pooled together and distributed evenly into new centrifuge tubes for single stain (and no stain) controls. These tubes are important for compensation of the flow c ytometer, as many of the fluorescent dyes used for staining (Step 16; Table 1) have overlapping emission spectra. The number of tubes taken for single staining depends on the number of fl uorescent c hannels being used for the experiment, whereas the amount of each sample taken to be pooled depends upon the number of cells in the sample and the number of cells expected to be positive for a given marker. When conducting a single stain analysis for a marker of a rare cell population, it is important to have enough cells to record a sufficient number of positive events and thereby enable adequate compensation of that fluorescent channel.
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15. Samples are centrifuged at 800×g for 4 min at 4°C to pellet cells. Supernatant is aspirated. 16. Samples are resuspended in at least 100 μl FACS buffer, into which is added a mixture of the relevant set of fluorescently labeled antibodies (e.g., Table 1) or each individual antibody for single stain controls (Step 14). Appropriate antibody dilutions should be determined empirically, although examples of dilutions for antibodies in the myeloid cell staining panel are given in Table 1. Samples are then incubated on ice in the dark for 30 min. 17. Samples are centrifuged at 800×g for 4 min at 4°C to pellet cells. Supernatant is aspirated. 18. (If necessary) If one or more of the labeling antibodies used in Step 16 is not directly conjugated to a fluorescent dye, an additional labeling step with a fluorescent secondary reagent is required. Samples are resuspended in FACS buffer, into which is added the appropriate dilution of the corresponding secondary reagent (e.g., anti-rat FITC for a rat primary antibody, streptavidin-PE for a biotinylated primary antibody), and then incubated on ice in the dark for 30 min. Samples are centrifuged at 800×g for 4 min at 4°C to pellet cells. Supernatant is aspirated. 19. (Recommended) Samples are resuspended in 100 μl 7-AAD Buffer (see below) at room temperature in the dark for 10 min. 7-AAD is a cell viability dye. Example of 1 ml preparation of 7-AAD buffer: 990 μl FACS buffer 10 μl 7-AAD staining solution (Table 1) 20. Sample volumes are brought up to no less than 200 μl with FACS buffer and transferred to a 5 ml FACS tube. For larger samples, more FACS buffer may be required to satisfy the cell acquisition requirements (see below). 21. Samples are acquired on flow cytometer, beginning with unstained and singly-stained cells so that fluorescence overlap between channels can be appropriately compensated, followed by cells stained with the complete antibody panel. Cells should not be acquired at a rate greater than 10,000 events/s, as this may lead to faulty acquisition and thus potentially compromise results. Once all samples have been acquired, each sample should be inspected individually to ensure that all compensation parameters are correct. Data can then be analyzed directly in FACSDiva or (recommended) exported and analyzed using
specialized software such as FlowJo or FCS Express.
Determination of Uterine Leukocyte Blood/Tissue Partitioning 1. Intravascular leukocyte labeling solution is freshly prepared (Section Equipment, Mice, and Supplies), stored on ice, and protected from light. 2. 100 μl of labeling solution is loaded into an insulin syringe and injected intravenously into the mouse. 3. 5 min following injection, mice are euthanized for tissue harvesting according to the institutionally approved protocol, and general leukocyte analysis (Section General Leukocyte Analysis) is performed.
Subset Analysis A gating strategy for identifying key leukocytes subsets in the myometrium and decidua is shown in Figure 1 and the relative abundance of these subsets on gd 9.5 is shown in Table 2. As discussed in the Figure 1 legend, the staining panel was optimized for the identification of myeloid cell types but also indirectly identifies lymphocytes. If the researcher intends to determine leukocyte blood/tissue partitioning as described in Section Determination of Uterine Leukocyte Blood/ Tissue Partitioning, the cells are first identified through the appropriate gating strategy and then plotted on the fluorescent channel corresponding to the fluorochrome used for in vivo labeling; cells that are positive in this channel are considered intravascular, whereas those that are negative are considered extravascular.
All Leukocytes RBCs (FSClo SSClo) and cell debris (FSClo SSChi) are excluded. Live leukocytes are identified as CD45+ 7-AAD−. Autofluorescent cells can be excluded using the open fluorescent channel (not depicted).
Macrophages The pregnant mouse uterus contains two subsets of macrophages that differ in their expression of MHCII and may represent a differentiation continuum. Macrophages are unique in their high expression of F4/80, although this marker can be expressed at lower levels on monocytes and eosinophils. Thus, macrophages are further delineated from monocytes based upon their lower expression
FLOW CYTOMETRY PROTOCOL CHAPTER 53
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Table 2 Identification and Relative Abundance of Leukocytes in E9.5 Implantation Sites (see Figure 1) Cell Type
Surface Phenotype
Myometrium
Decidua
Neutrophils
Gr-1hi Ly6Cint
1.2%
22.4%
MHCII− monocytes
Ly6Chi (Gr-1+)a MHCII−
13.7%
23.4%
MHCII+
Ly6Chi
monocytes
(Gr-1+)a
MHCII+
14.1%
13.0%
MHCIIlo macrophages
Ly6Cneg-int F4/80+ MHCIIlo
21.1%
3.1%
MHCIIhi macrophages
Ly6Cneg-int F4/80+ MHCIIhi
15.8%
4.9%
CD11blo dendritic cells (DCs)
Ly6Cneg-int F4/80− MHCIIhi CD11chi CD11blo (CD103+)a
2.9%
0.4%
CD11bhi
Ly6Cneg-int
DCs
F4/80−
MHCIIhi
CD11chi
CD11bhi
(CD103−)a
2.3%
0.8%
Eosinophils
Ly6Cneg-int F4/80lo SSChi
2.9%
N.D.
Natural killer cells
Ly6Cneg-int F4/80− CD11b+ CD11c+ (indirect)a
9.6%
7.6%
T cells
Ly6Cneg-int F4/80− CD11b− CD11c− (indirect)a
5.8%
6.4%
89.5%
82.1%
Total identified N.D., not detectable. aSee Figure 1 legend for further explanation.
of Ly6C and from eosinophils based on the fact that eosinophils are uniquely FSClo SSChi. Importantly, F4/80 should be placed on one of the brighter fluorescent channels (i.e., PE, PE-Cy7, or APC) because overall expression of this marker is relatively low even on macrophages.
Monocytes Blood monocytes that have newly emerged from the bone marrow have a Ly6Chi surface phenotype and are thought to return to bone marrow to become Ly6Clo monocytes.8 Ly6Chi monocytes are abundant in implantation sites up until ∼gd 10.5, but extravasate rapidly only in the myometrium, where they differentiate into tissue macrophages. In contrast, they primarily remain intravascular in the decidua, where they show moderately downregulated Ly6C expression by gd 9.5. This downregulation can complicate their discrimination from macrophages. Like macrophages, Ly6Chi monocytes can be divided into two subsets based upon MHCII expression.
Neutrophils Neutrophils are readily detected in any mouse tissue based upon their high expression of Ly6G. They can also be identified with Gr-1 antibodies, which bind strongly to Ly6G and weakly to Ly6C; neutrophils thus appear Gr-1hi and Ly6Chi monocytes appear Gr-1lo. Importantly, due to the high expression of Gr-1 and Ly6C on neutrophils and monocytes, respectively, either of these markers can be placed on Alexa Fluor 700, the dimmest of the available fluorescent channels (Table 1). Although neutrophils constitute a major population in the decidua, as with monocytes,
many of the cells are intravascular and are bound to endothelial cells within the vascular zone.9
Dendritic Cells All DCs highly express MHCII and are differentiated from other tissue-resident APCs, such as macrophages and B cells, by their high expression of CD11c (B Cells are CD11c−) and low-to-absent expression level of F4/80 (macrophages are F4/80+). The pregnant mouse uterus harbors two subsets of DCs that differ based upon their expression of CD103 and CD11b.10
Eosinophils Eosinophils (Figure 1) are abundant in the murine nonpregnant uterus during estrus, but during pregnancy are only present within the myometrium (Table 2). They express intermediate levels of F4/80 and can be differentiated from other myeloid cells based on their uniquely high SSC properties.
Natural Killer Cells Using the staining panel optimized for myeloid cell types (Table 1), uNK cells can be indirectly identified as CD45+ cells that fail to express Ly6C, MHCII, or F4/80, but express both CD11b and CD11c.11 More directly, uNK cells are identified as CD3−CD122+.3 They can then be divided into subsets through the use of DX5 antibodies (or NK1.1 antibodies in certain mouse strains, namely C57BL/6, FVB/N, NZB, NZW, and SJL) and fluorescently labeled DBA lectin (see Chapter 19).
T Cells Using the staining panel optimized for myeloid cell types (Table 1), T cells in the pregnant mouse uterus
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can be indirectly identified as CD45+ cells that fail to express any of the other markers. More directly, they are identified using CD3 antibodies, and can be further subsetted using antibodies to TCRβ and TCRγ/δ (to differentiate αβ and γδ T cells), CD4 and CD8 to distinguish αβ T cell subsets, and by intracellular staining for select cytokines and FOXP3 to distinguish different CD4 T cell subsets. NKT cells are present at low numbers and mostly can be identified as CD3+ DX5+ or, in the appropriate mouse strains, as CD3+ NK1.1+ (see Section Natural Killer Cells).12
B Cells B cells are rare in nonpregnant and pregnant uteri under noninflammatory conditions.13 In principle, they can be identified as Ig+ MHCII+ CD19+.
Technical Limitations Although flow cytometry offers a highly sensitive and specific means to visualize all leukocyte subsets within the pregnant mouse uterus, it cannot provide information regarding the position of these cells in situ aside from a basic discrimination between the myometrium and decidua. Such information requires tissue immunostaining, as described in Chapter 49. Furthermore, analysis of a given uterine tissue layer will not be able to distinguish intravascular from extravascular leukocytes unless a specific technique (Section Determination of Uterine Leukocyte Blood/Tissue Partitioning) for this discrimination is employed.
the problem of placental contamination of decidual tissue has already been discussed, it should also be noted that the placenta contains fetal leukocytes, many of which are autofluorescent and can be gated away if one fluorescent channel is left open (Table 1). Finally, the availability of a three-laser (10-color) flow cytometer is not always guaranteed. We recommend the use of 10 colors for the identification of leukocyte populations when possible, but 6-color or 3-color formats can be used for more basic analyses.
References 1. Tagliani E, Shi C, Nancy P, Tay CS, Pamer EG, Erlebacher A. Coordinate regulation of tissue macrophage and dendritic cell population dynamics by CSF-1. J Exp Med August 29, 2011;208(9):1901–16. 2. Movahedi K, Laoui D, Gysemans C, Baeten M, Stange G, Van den Bossche J, et al. Different tumor microenvironments contain functionally distinct subsets of macrophages derived from Ly6C(high) monocytes. Cancer Res July 15, 2010;70(14):5728–39. 3. Yadi H, Burke S, Madeja Z, Hemberger M, Moffett A, Colucci F. Unique receptor repertoire in mouse uterine NK cells. J Immunol November 1, 2008;181(9):6140–7. 4. Fridman WH, Galon J, Pages F, Tartour E, Sautes-Fridman C, Kroemer G. Prognostic and predictive impact of intra- and peritumoral immune infiltrates. Cancer Res September 1, 2011;71(17):5601–5. 5. Kruse A, Merchant MJ, Hallmann R, Butcher EC. Evidence of specialized leukocyte-vascular homing interactions at the maternal/fetal interface. Eur J Immunol April 1999;29(4):1116–26. 6. Welsh AO, Enders AC. Light and electron microscopic examination of the mature decidual cells of the rat with emphasis on the antimesometrial decidua and its degeneration. Am J Anat January 1985;172(1):1–29. 7. Nancy P, Tagliani E, Tay CS, Asp P, Levy DE, Erlebacher A. Chemokine gene silencing in decidual stromal cells limits T cell access to the maternal-fetal interface. Science June 8, 2012;336(6086):1317–21. 8. Geissmann F, Manz MG, Jung S, Sieweke MH, Merad M, Ley K. evelopment of monocytes, macrophages, and dendritic cells. Science D February 5, 2010;327(5966):656–61.
Importantly, different cell subsets have varying capacities to survive enzymatic tissue digestion and the shear stress that accompanies cell acquisition. In particular, uNK cells, epithelial cells, and stromal cells are large and fragile; therefore, they are more susceptible to cell death. These subsets might consequently be underrepresented in FACS analyses. Certain cell preparation protocols have been developed to better preserve NK cells.3
9. Karsten CM, Kruse A. The role of vascular addressins in implantations sites during successful and failing mouse pregnancies. Immunol Invest 2008;37(5):449–66.
Another issue that can arise starting at gd 10.5 is placental contamination of decidual tissue (see Section General Leukocyte Analysis; Step 3). Although
13. Cha HR, Ko HJ, Kim ED, Chang SY, Seo SU, Cuburu N, et al. Mucosa-associated epithelial chemokine/CCL28 expression in the uterus attracts CCR10+ IgA plasma cells following mucosal vaccination via estrogen control. J Immunol September 15, 2011;187(6):3044–52.
10. Collins MK, Tay CS, Erlebacher A. Dendritic cell entrapment within the pregnant uterus inhibits immune surveillance of the maternal/fetal interface in mice. J Clin Invest July 2009;119(7):2062–73. 11. Mallidi TV, Craig LE, Schloemann SR, Riley JK. Murine endometrial and decidual NK1.1+ natural killer cells display a B220+CD11c+ cell surface phenotype. Biol Reprod August 2009;81(2):310–8. 12. Boyson JE, Aktan I, Barkhuff DA, Chant A. NKT cells at the maternal-fetal interface. Immunol Invest 2008;37(5):565–82.
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Adam Blaisdell Department of Pathology, NYU School of Medicine, NY, USA
Adrian Erlebacher Department of Pathology, NYU School of Medicine, NY, USA; NYU Cancer Institute, NYU School of Medicine, NY, USA CHAPTER SUMMARY Flow cytometry is a powerful tool with single-cell resolution that allows the researcher to gain information on the cellular composition of a tissue and to analyze multiple cell subsets at once. Here, we provide a methodological framework for applying flow cytometry to the study of leukocyte biology in the pregnant mouse uterus.
Abbreviations
PBS
phosphate buffered saline
7-AAD
7-aminoactinomycin D
PE phycoerythrin
ACK
ammonium chloride potassium
PES polyethersulfone
APC allophycocyanin
RBC
red blood cell (erythrocyte)
BrdU bromodeoxyuridine
RT
room temperature
SSC
side scatter
uNK
uterine natural killer
BSA
bovine serum albumin
Cy7
cyanine 7
ddH2O
double-distilled water
DBA
Dolichos biflorus lectin
DC
dendritic cell
FACS
fluorescence-activated cell sorting
FBS
fetal bovine serum
FITC
fluorescein isothiocyanate
FSC
forward scatter
HBSS
hank’s balanced salt solution
MHCII
class II major histocompatibility complex, which presents peptide antigens to CD4 T cells
Overview Many hematopoietic and nonhematopoietic cell types reside within peripheral tissues, and the pregnant mouse uterus is no exception. Multicolor flow cytometry offers a powerful tool to simultaneously analyze all of these cell populations using combinations of fluorescently labeled antibodies directed against different surface antigens. This technique is the analytic version of fluorescence-activated cell sorting (FACS), which is used to purify live cells. In this chapter, we provide a methodological framework for applying flow cytometry to the study of uterine leukocytes, with emphasis on approaches for dividing the cells
The Guide to Investigation of Mouse Pregnancy. http://dx.doi.org/10.1016/B978-0-12-394445-0.00053-9 © 2014 Elsevier Inc. All rights reserved.
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(A) SSC
7-AAD
Viable leukocytes
FSC
MHCII+ monocytes
CD45
MHCII
CD11c
CD11blo DCs CD11bhi DCs
MHCII– monocytes
CD11b NK cells
ev I iv MHCIIhi macrophages MHCIIlo macrophages
MHCII
F4/80
Ly6C
FSC
MHCII
MHCII
Gr-1
SSC
Neutrophils
ev I iv
F4/80
FSC
CD11b
SSC
T cells
SSC
SSC
CD11c
Eosinophils
FSC
FSC
(B) SSC
7-AAD
Viable leukocytes
FSC
MHCIIhi monocytes
CD45
MHCII
CD11c
CD11blo DCs CD11bhi DCs
F4/80
FSC
T cells CD11b
ev I iv MHCIIhi macrophages MHCIIlo macrophages ev I iv
SSC
SSC
CD11c
CD11b NK cells
MHCIIlo monocytes
MHCII
F4/80
Ly6C
FSC
MHCII
MHCII
Gr-1
SSC
Neutrophils
FSC
Figure 1 Flow cytometric identification of leukocytes in gd 9.5 implantation sites of pregnant C57BL/6 mice. The myometrium (A) was dissected away from the decidua (B) prior to tissue digestion and analysis by a 3-laser, 10-color BD LSRII flow cytometry. The figure is meant to demonstrate a general gating scheme that allows one to identify as many different important leukocytes as possible, with emphasis on myeloid cells. Alternate gating schemes, additional criteria using the markers shown, and the use of additional markers (as permitted by the number of available fluorescence channels) will allow for greater specificity. For example, it is not appropriate to rely upon this gating strategy to identify T cells and NK cells because these cells should be identified using antibodies to CD3, TCRβ, and CD122. The gating scheme shown employed the following antibody-fluorochrome conjugates: CD45-Pacific orange; Ly6C-APC; Gr-1-Alexa 700; F4/80-FITC; MHCII-Pacific blue; CD11b-PE; CD11c-PE-Cy7. The PE-Texas Red and APC-Cy7 channels were not used. CD45-Pacific Orange and Gr-1-Alexa 700 are preferred conjugates because their corresponding fluorescence channels (AmCyan and Alexa 700, respectively) are dim. Summary of the gating strategy: After a broad gating on FSC vs SSC to include all possible leukocytes, viable leukocytes are identified as CD45+ 7-AAD− cells. Neutrophils are then identified as Gr-1hi Ly6Cint cells, which greatly simplifies the subsequent specific visualization of Ly6Chi monocytes. Because of the dimness of Alexa 700 fluorochrome, the neutrophils
FLOW CYTOMETRY PROTOCOL CHAPTER 53
into their major subsets. We will not provide a comprehensive overview of flow cytometry because this can be found elsewhere in many books (e.g., Current Protocols in Immunology). Flow cytometry can also be used to determine expression levels of intracellular proteins, DNA content, and proliferation status. However, these techniques are also outside the scope of this chapter and are discussed in detail elsewhere. The importance of flow cytometry in studying uterine leukocyte biology during pregnancy is becoming increasingly evident. Most strikingly, the technique allows for the direct visualization, and thus the discovery, of uterine leukocyte subsets. For example, we recently used this technique to show that uterine macrophages divide into two subsets based upon their surface expression levels of class II major histocompatibility complex, which presents peptide antigens to CD4 T cells (MHCII).1 These two subsets show differential expression of several genes involved in tissue remodeling, thus suggesting they may have unique nonredundant roles in the pregnant uterus.1,2 Flow cytometry has also uncovered two subsets of uterine natural killer (uNK) cells within the pregnant mouse uterus that share expression of the interleukin-2 receptor β subunit (CD122) but differ in their binding to Dolichos biflorus agglutinin (DBA).3 As with the aforementioned macrophages, these uNK cells show distinct expression profiles and may therefore execute different functions during pregnancy. When combined with measurements of tissue weight, flow cytometry can also be used to calculate both leukocyte tissue densities within uterine tissue layers as well as absolute leukocyte number per implantation site. Density, rather than absolute number, likely has the greater functional relevance. The importance of this distinction can best be exemplified by the correlation of certain leukocyte densities
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with prognosis in growing tumors4 and likely holds true for rapidly growing tissues, such as the myometrium and decidua. Indeed, macrophage densities in the myometrium remain relatively constant throughout pregnancy, despite the fact that their absolute numbers per implantation site increase drastically; conversely, decidual macrophage densities drop considerably without a significant change in absolute number per implantation site.1 It is therefore likely that the decidua has mechanisms in place to control macrophage densities (as opposed to absolute number), possibly in an effort to minimize the occurrence of an adverse inflammatory reaction that could compromise the health of the fetus. Flow cytometry can also be used to discriminate intravascular vs extravascular leukocytes (Section Determination of Uterine Leukocyte Blood/Tissue Partitioning).1 This turns out to be an important distinction in the context of the pregnant mouse uterus because several leukocyte subsets (e.g., monocytes) show considerably lower rates of extravasation into the decidua as compared to the myometrium under steady-state conditions. As a result, the decidua contains a large number of monocytes within its intravascular compartment, whereas myometrial monocytes are mainly extravascular (see Figure 1). The technique we describe here to reveal leukocyte blood/tissue partitioning involves intravenous injection of a low dose of pan-leukocyte (anti-CD45) antibodies several minutes prior to sacrifice, thus allowing for the select labeling of all intravascular cells. This technique provides a distinct advantage over vascular perfusion because some decidual leukocytes, including monocytes, remain adherent to the decidual vasculature even after perfusion.1,5,6 The flow cytometric analysis of peripheral tissue leukocytes requires the preparation of single-cell
here appear simply as Gr-1+, while Ly6Chi monocytes appear Gr-1− even though they express this marker at fairly high levels. Ly6Chi monocytes divide into MHCII− and MHCII+ subsets. In the myometrium, Ly6Chi monocytes are predominantly extravascular. This is particularly the case for MHCII+Ly6Chi monocytes, which also show some F4/80 upregulation presumably reflecting an initial step in their differentiation into macrophages. In contrast, Ly6Chi monocytes in the decidua are predominantly intravascular, and appear to have undertaken a differentiation program of MHCII and F4/80 upregulation even within the intravascular compartment. Macrophages, which are Ly6Cneg-intF4/80+ cells and divide into MHCIIlo and MHCIIhi subsets, are extravascular in both the myometrium and decidua. DCs are identified as Ly6Cneg-intMHCII+F4/80−CD11chi cells that divide into CD11blo (CD103+; not shown) and CD11bhi (CD103−; not shown) subsets. We prefer a stringent CD11chi gate for CD11bhi DCs to exclude as many macrophages as possible because the prior MHCII vs F4/80 gate used to collectively identify DCs will not exclude all macrophages. Eosinophils are F4/80lo cells that show a characteristic high SSC profile; note that these cells are absent from the decidua. After excluding all the myeloid populations listed above, uNK cells are within the remaining CD11b+ CD11c+ population; T cells are within the CD11b−CD11c− population. Note that uNK cells are more granular than T cells. MHCII+F4/80−CD11b−CD11c− cells in the decidua are probably B cells. In a separate experiment, CD45-PE-Cy7 antibodies (1 μg) were intravenously injected 5 min before sacrifice in order to discriminate intravascular (iv) from extravascular (ev) macrophages and monocytes. These plots used a different set of antibody-fluorochrome conjugates for cell identification (not shown).
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suspensions. This typically involves a step of limited proteolysis to disrupt cell–cell interactions and to release leukocytes from their attachments to the extracellular matrix. This treatment, however, must maintain the integrity of all surface antigens of interest. To this end, Liberase TM Research Grade (Roche) has proven most effective in our hands. Within 30 min, both myometrium and decidua can be reduced to a virtual single-cell suspension; to our knowledge, no relevant epitopes have been compromised. If the analysis requires a complete analysis of nonleukocytes as well, trypsin can be added to the Liberase cocktail to allow complete release of stromal and endothelial cells. Trypsin, however, destroys many surface epitopes, thus limiting the discrimination of many cell types and in-depth analysis of surface phenotypes. However, we have found that trypsin does not destroy the epitopes recognized by antibodies directed against CD45, ICAM-2 (CD102), and EpCAM (CD326), thus allowing the broad discrimination of leukocytes (CD45+), endothelial cells (CD45− CD102+ CD326−), epithelial cells (CD45− CD102+ CD326+), and stromal cells (CD45− CD102− CD326−).7 Following data collection, the typical first step of a flow cytometric analysis is to display the cells on a side scatter (SSC; a measure of cell granularity) vs forward scatter (FSC; a measure of cell size) plot (Figure 1). This allows the researcher not only to gate away cell debris and red blood cells (RBCs), but also to enrich for leukocytes because they tend to be smaller than
nonleukocytes. However, we have found it to be extremely useful to include the panleukocyte marker CD45 in our staining cocktail in order to definitively identify leukocytes and avoid the analytic complications that otherwise arise when using certain markers (e.g., Ly6C) that are expressed by both leukocytes and nonleukocytes. We typically use an anti-CD45 Pacific Orange conjugate (Table 1) because the corresponding fluorescent channel (AmCyan) is quite dim and thus unusable for many other markers. We also use a live-dead discrimination reagent (e.g., propidium iodide or, preferably, 7-aminoactinomycin D (7-AAD)) in order to gate away dead cells, which can be autofluorescent and thus appear to express many surface markers. Finally, if density analyses are performed, it is important to maintain consistency in cell preparation and total live cell determination in order to be able to combine results from multiple experimental runs. We determine the total number of live cells in our suspensions using a hemocytometer and trypan blue exclusion. Finally, one fluorescent channel (e.g., PE-Texas Red, which is otherwise not useful) can be left open to allow for identification of naturally autofluorescent cells. In the late-gestation pregnant mouse uterus, autofluorescent cells appear as a wisp emanating from the CD45+ population. These cells are primarily of fetal origin and should be gated away, thereby preserving an exclusive analysis of maternal leukocytes. Given the above basic principles, more specific analytic strategies can be developed, depending
Table 1 Example of Staining Panel Used to Identify Myeloid Cells in Myometrial and Decidual Tissue as well as to Discriminate Intravascular vs Extravascular Leukocytes Fluorochromea
Epitope
Supplier
Clone
Catalog Number
Dilution
Fluorescein isothiocyanate
Ly6C
Biolegend
HK1.4
128005
1:400
Phycoerythrin (PE)
F4/80
Biolegend
BM8
123109
1:100
PE-Texas Redb
–
–
–
–
–
7-AAD (PerCP-Cy5.5)
–
BD Pharmingen
–
51-68981E
1:100
PE-Cy7
CD45 (intravascular)
Biolegend
30-F11
103113
–
Pacific blue
MHCII (I-A/I-E)
Biolegend
M5/114.15.2
107619
1:400
Pacific orange (AmCyan)
CD45 (total)
Invitrogen
30-F11
MCD4530
1:100
Allophycocyanin (APC)
CD11c
Biolegend
N418
117309
1:200
Alexa 700
Gr-1c
Biolegend
RB6-8C5
108421
1:200
APC-Cy7
CD11b
Biolegend
M1/70
101225
1:200
aBased
upon fluorescent channels available on 3-laser (violet: 405 nm; blue: 488 nm; red: 633 nm) 10-color LSRII flow cytometer (BD Bioscience). fluorescent channel left open to identify autofluorescent cells. cCan be replaced with anti-Ly6G antibodies, clone 1A8. bThis
FLOW CYTOMETRY PROTOCOL CHAPTER 53
upon whether the user wishes to analyze many cell subsets at once or perform an in-depth surface phenotype analysis of one specific subset. The most sophisticated flow cytometry currently available can track up to 18 different markers. For most analyses, a 10-color format based on the use of a three-laser flow cytometer—violet (405 nm), blue (488 nm), and red (633 nm)—suffices for identification of all cell subsets within the mouse uterus, and the protocols and schema described herein are based upon this format. Indeed, using 10-color flow cytometry, virtually all cells of myeloid origin (macrophages, dendritic cells (DCs), monocytes, neutrophils, eosinophils) can be identified in one staining scheme (Table 1, Figure 1), and a similar approach can be used for cells of lymphoid origin. However, any single population can typically be identified with fewer than 10 markers, which thus allows for further phenotypic characterization using the open fluorescent channels.
Protocol Equipment, Mice, and Supplies 1. Equipment a. Flow cytometer, LSRII (BD Biosciences), three-lasers (405, 488, 633 nm) b. Cell acquisition software (BD Biosciences FACSDiva) c. FACS data analysis software, such as FlowJo (Tree Star) or FCS Express (De Novo Software) d. Microcentrifuge, refrigerated e. Water bath, 37°C 2. Dissection microscope and light source 3. Instruments a. Dissection/mincing tools Scissors, straight, 14.5 cm (Fine Science Tools #14000-14) Scissors, extra narrow, 10.5 cm (Fine Science Tools #14088-10) Scissors, fine, curved, 10.5 cm (Fine Science Tools #14061-10) Graefe forceps, straight, 10 cm (Fine Science Tools #11050-10) Dumont #5 forceps, straight, 11 cm (Fine Science Tools #11251-20) b. Pipetters (1 ml, 100 μl, 10 μl) with tips c. 10 cm Petri dish (BD Falcon #351029) d. 1.7 ml microcentrifuge tubes (Denville Scientific #C2170)
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e. Sterile disposable filter, polyethersulfone (PES) membrane (90 mm), 0.2 μm pore size (Thermo Scientific #569-0020) f. Cell strainer, nylon mesh, 70 μm (BD Falcon #352350) g. Hematocytometer and light microscope h. 5 ml FACS tube, polystyrene, round bottom (BD Falcon #352052) i. (Optional) Insulin syringe (Becton Dickinson #309301) 4. Nonpregnant or pregnant mice, or mice with artificial deciduas 5. Reagents a. Hanks’ Balanced Salt Solution (HBSS) with Ca2+ and Mg2+ (Invitrogen #14025-076) b. Liberase TM Research Grade (Roche #05401127001), diluted to 28 Wunsch units (WU)/ml (100×) in double-distilled water (ddH2O) and stored in 50 or 100 μl aliquots at −20°C; thawed as needed and used immediately c. DNase I (Roche #10104159001), diluted to 30 mg/ml (1000×) in ddH2O and stored in 5–10 μl aliquots at −20°C; thawed as needed and used immediately d. (Optional) Trypsin, 2.5%, no phenol red (Invitrogen #15090-046, 50×) stored in 100 or 200 μl aliquots at −20°C; thawed as needed and used immediately e. (Optional) Ammonium chloride potassium (ACK) lysing buffer (Lonza #10-548E) f. Phosphate-buffered saline (PBS) without Ca2+ and Mg2+ (Cellgro #21-031-CV) g. Bovine serum albumin (BSA; Sigma #A2153) h. Trypan Blue (Thermo Scientific #SV30084.01) i. FcγR blocking antibody (anti-CD16/32; Clone 2.4G2). This reagent is available from multiple vendors. We buy ours in bulk from BioXCell (#BE0008). The antibody is aliquoted at 1 mg/tube and stored at −80°C until needed, then diluted to 1 mg/ml in PBS and stored at 4°C j. (Optional) RPMI 1640 medium (Invitrogen #21870-076) k. (Optional) Fetal bovine serum (FBS; Atlanta Biologicals #S12450), heat inactivated for 30 min at 55°C l. (Optional) Heparin sodium, 1000 USP units/ ml (Sagent Pharmaceuticals #400-01) 6. Solutions
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a. Tissue digestion buffer (example of 10 ml preparation) 9.89 ml HBSS containing Ca2+ and Mg2+ 100 μl Liberase TM (28 WU/ml); final concentration 0.28 WU/ml 10 μl DNase I (30 mg/ml); final concentration 30 μg/ml (Optional) 200 μl Trypsin (2.5%); final w:v 0.05%; subtract from HBSS volume accordingly b. FACS buffer (example of 500 ml preparation) 495 ml PBS without Ca2+ and Mg2+ 5 g BSA; final w:v 1% 5 ml 0.5 M EDTA, pH 8.0; final concentration 5 mM Filter buffer through PES membrane with 0.2 μm pore size prior to use in order to sterilize and degas. c. (Optional) RPMI-10 (example of 500 ml preparation) 450 ml RPMI 1640 medium 50 ml FBS Filter buffer through PES membrane with 0.2 μm pore size prior to use. d. (Optional) Intravascular leukocyte labeling solution (100 μl per mouse) 90 μl PBS without Ca2+ and Mg2+ 5 μl Heparin sodium, 5 USP units total 5 μl anti-mouse CD45 (Clone 30-F11, 0.2 mg/ml), 1 μg total, preferably conjugated to a brighter fluorochrome such as PE (Biolegend #103105), PE-Cy7 (Biolegend #103113), or allophycocyanin (APC; Biolegend #103111)
Methods General Leukocyte Analysis
1. Tissue digestion buffer is freshly prepared (see Section Equipment, Mice, and Supplies) and stored on ice. 2. Mice are euthanized according to the institutionally approved protocol. Uteri are immediately dissected: fine straight dissection scissors are used to sever the cervical attachment, after which the cervix is held with Graefe forceps in a raised position, allowing the mesometrium to be peeled away with Dumont no. 5 forceps; both fallopian tube attachments are then severed to completely release the uterus, which is then immersed in cold HBSS within a 10 cm Petri dish.
3. Using the dissection microscope and light source, the mesometrium is trimmed off the uterus, and the horns are divided at the cervix. Individual implantation sites are then separated from each other by cutting at the point where the interimplantation site directly abuts the decidua; the interimplantation sites are then d iscarded unless they are to be analyzed in parallel with the myometrium and decidua. For gestation day (gd) 4.5 and gd 5.5, the myometrium and decidua cannot be separated, and these tissues are thus analyzed together (see Chapter 1). By gd 6.5, however, the myometrium can easily be separated from underlying decidua, as these tissues are only tightly attached at the mesometrial pole. The free edge of the myometrium is grasped with Dumont no. 5 forceps at the point where the interimplantation site was severed, and snipped once with fine scissors to increase the size of the hole (see Chapter 2). The myometrium is then peeled away from the decidua by threading it through Graefe forceps. The embryo and yolk sac are removed from the interior of the decidua using Dumont no. 5 forceps. This procedure is performed for each implantation site separately. At gd 11.5 and later, the placenta will comprise a significant fraction of the dissected “decidual” tissue. Although it is possible to separate decidua from placenta, this process is difficult, time-consuming, and imprecise. Therefore, after discarding the fetus, its amniotic and yolk sac membranes and the umbilical cord, the decidua, and placenta are taken together for further analysis. The decidual localization of a cell type of interest, however, needs to be confirmed by tissue immunostaining. 4. The myometrium and decidua (or combined decidual/placental tissue) from each litter are separately pooled into 1 ml tissue digestion buffer within a microcentrifuge tube at no more than 100 mg tissue/ml. If the amount of t issue exceeds 100 mg, multiple microcentrifuge tubes are prepared to accommodate the excess. Using an insufficient amount of tissue digestion buffer will lead to incomplete digestion, which could compromise the results of the e xperiment. If necessary to study rare cell populations, specimens from multiple litters can also be pooled. 5. Tissues are minced within the microcentrifuge tube with fine curved dissection scissors until large chunks are no longer visible.
FLOW CYTOMETRY PROTOCOL CHAPTER 53
6. Samples are incubated in a water bath at 37°C for 45 min. After the first 15 min, and then every 5–10 min thereafter, the tissue fragments are disaggregated by pipetting the solution m ultiple times through a 1 ml pipet tip. Ultimately the solution should resemble a soup and should be easily pipetted. If not, the tubes can be incubated for an additional 15 min at 37°C. Special consideration: If trypsin is additionally added to the tissue digestion buffer in order to generate single-cell suspensions of stromal cells and endothelial cells, the incubation time should be increased to 60 min. EDTA (to 5 mM final concentration) should be added for the last 15 min in order to inhibit Ca2+- and Mg2+-dependent adhesive interactions, with the solution again pipetted every 5–10 min. 7. Samples are centrifuged at 1000×g for 4 min at 4°C to pellet cells. Supernatant is aspirated. 8. (Optional; recommended for decidua) Although abundant, RBCs are not an interesting cell population within the uterus and can therefore be lysed in an effort to “purify” all other cells and thus speed data acquisition on the flow cytometer. RBC lysis is especially important for the blood-rich decidua. Samples are r esuspended in 1 ml ACK lysing buffer and promptly transferred to a 15-ml conical tube. Then, 9 ml of ice-cold RPMI-10 is immediately added to stop the lysing reaction. Samples are centrifuged at 1000×g for 4 min at 4°C to pellet cells. Supernatant is aspirated, and the resuspended pellet is washed again with 10 ml RPMI-10. If it is observed that a significant number of RBCs remain after lysis, the lysing reaction can be continued for a longer time before the addition of RPMI-10. However, the lysing reaction can potentially destroy leukocytes and nonleukocytes. Thus, it should be performed on ice and not allowed to continue for longer than 5 min. 9. Samples are resuspended in 1 ml FACS buffer and incubated in a water bath at 37°C for 15 min, and then pipetted several times though a 1-ml pipet tip. This step serves to further disaggregate the cells by inhibiting Ca2+- and Mg2+dependent adhesive interactions. 10. Samples are filtered through a 70-μm nylon mesh cell strainer into a new microcentrifuge tube. 11. Samples are centrifuged at 800×g for 4 min at 4°C to pellet cells. Supernatant is aspirated. 12. Samples are resuspended in 1 ml FACS Buffer and 10 μl is taken for a cell count. Samples are
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centrifuged at 800×g for 4 min at 4°C to pellet cells. Supernatant is aspirated. Although cell counts are essential for cell density analysis, they should be performed for all experimental formats to ensure optimal labeling of cells with antibodies (Steps 13, 16). 13. Samples are resuspended in Fc blocking buffer (see below) at a concentration no greater than 106 cells per 100 μl buffer and incubated on ice for 10 min. Even if there are fewer than 106 cells in a given sample, a minimum of 100 μl buffer should be used. For both the Fc blocking and labeling steps (Steps 16, 19), it is important to keep a relatively low cell density in order to sufficiently block and label all cells. If the n umber of cells in a given sample greatly exceeds 106, one of two options can be chosen: (1) A smaller aliquot of the sample (<106 cells) is taken and used to represent the entire sample. This option is useful when analyzing cell subsets that are relatively abundant within the tissue; therefore, only a small number of cells will need to be acquired by the flow cytometer. (2) A larger amount of Fc blocking buffer (and staining b uffer in Step 16) can be added to the entire sample to maintain a relatively low cell density. This option is useful when analyzing rare cell subsets that require acquisition of a large number of total cells. Example of 1 ml preparation of Fc blocking buffer: 990 μl FACS buffer 10 μl FcγR blocking antibody (1 mg/ml); final concentration 10 μg/ml 14. Samples are brought to 1 ml with FACS buffer. Small amounts of each sample are pooled together and distributed evenly into new centrifuge tubes for single stain (and no stain) controls. These tubes are important for compensation of the flow c ytometer, as many of the fluorescent dyes used for staining (Step 16; Table 1) have overlapping emission spectra. The number of tubes taken for single staining depends on the number of fl uorescent c hannels being used for the experiment, whereas the amount of each sample taken to be pooled depends upon the number of cells in the sample and the number of cells expected to be positive for a given marker. When conducting a single stain analysis for a marker of a rare cell population, it is important to have enough cells to record a sufficient number of positive events and thereby enable adequate compensation of that fluorescent channel.
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15. Samples are centrifuged at 800×g for 4 min at 4°C to pellet cells. Supernatant is aspirated. 16. Samples are resuspended in at least 100 μl FACS buffer, into which is added a mixture of the relevant set of fluorescently labeled antibodies (e.g., Table 1) or each individual antibody for single stain controls (Step 14). Appropriate antibody dilutions should be determined empirically, although examples of dilutions for antibodies in the myeloid cell staining panel are given in Table 1. Samples are then incubated on ice in the dark for 30 min. 17. Samples are centrifuged at 800×g for 4 min at 4°C to pellet cells. Supernatant is aspirated. 18. (If necessary) If one or more of the labeling antibodies used in Step 16 is not directly conjugated to a fluorescent dye, an additional labeling step with a fluorescent secondary reagent is required. Samples are resuspended in FACS buffer, into which is added the appropriate dilution of the corresponding secondary reagent (e.g., anti-rat FITC for a rat primary antibody, streptavidin-PE for a biotinylated primary antibody), and then incubated on ice in the dark for 30 min. Samples are centrifuged at 800×g for 4 min at 4°C to pellet cells. Supernatant is aspirated. 19. (Recommended) Samples are resuspended in 100 μl 7-AAD Buffer (see below) at room temperature in the dark for 10 min. 7-AAD is a cell viability dye. Example of 1 ml preparation of 7-AAD buffer: 990 μl FACS buffer 10 μl 7-AAD staining solution (Table 1) 20. Sample volumes are brought up to no less than 200 μl with FACS buffer and transferred to a 5 ml FACS tube. For larger samples, more FACS buffer may be required to satisfy the cell acquisition requirements (see below). 21. Samples are acquired on flow cytometer, beginning with unstained and singly-stained cells so that fluorescence overlap between channels can be appropriately compensated, followed by cells stained with the complete antibody panel. Cells should not be acquired at a rate greater than 10,000 events/s, as this may lead to faulty acquisition and thus potentially compromise results. Once all samples have been acquired, each sample should be inspected individually to ensure that all compensation parameters are correct. Data can then be analyzed directly in FACSDiva or (recommended) exported and analyzed using
specialized software such as FlowJo or FCS Express.
Determination of Uterine Leukocyte Blood/Tissue Partitioning 1. Intravascular leukocyte labeling solution is freshly prepared (Section Equipment, Mice, and Supplies), stored on ice, and protected from light. 2. 100 μl of labeling solution is loaded into an insulin syringe and injected intravenously into the mouse. 3. 5 min following injection, mice are euthanized for tissue harvesting according to the institutionally approved protocol, and general leukocyte analysis (Section General Leukocyte Analysis) is performed.
Subset Analysis A gating strategy for identifying key leukocytes subsets in the myometrium and decidua is shown in Figure 1 and the relative abundance of these subsets on gd 9.5 is shown in Table 2. As discussed in the Figure 1 legend, the staining panel was optimized for the identification of myeloid cell types but also indirectly identifies lymphocytes. If the researcher intends to determine leukocyte blood/tissue partitioning as described in Section Determination of Uterine Leukocyte Blood/ Tissue Partitioning, the cells are first identified through the appropriate gating strategy and then plotted on the fluorescent channel corresponding to the fluorochrome used for in vivo labeling; cells that are positive in this channel are considered intravascular, whereas those that are negative are considered extravascular.
All Leukocytes RBCs (FSClo SSClo) and cell debris (FSClo SSChi) are excluded. Live leukocytes are identified as CD45+ 7-AAD−. Autofluorescent cells can be excluded using the open fluorescent channel (not depicted).
Macrophages The pregnant mouse uterus contains two subsets of macrophages that differ in their expression of MHCII and may represent a differentiation continuum. Macrophages are unique in their high expression of F4/80, although this marker can be expressed at lower levels on monocytes and eosinophils. Thus, macrophages are further delineated from monocytes based upon their lower expression
FLOW CYTOMETRY PROTOCOL CHAPTER 53
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Table 2 Identification and Relative Abundance of Leukocytes in E9.5 Implantation Sites (see Figure 1) Cell Type
Surface Phenotype
Myometrium
Decidua
Neutrophils
Gr-1hi Ly6Cint
1.2%
22.4%
MHCII− monocytes
Ly6Chi (Gr-1+)a MHCII−
13.7%
23.4%
MHCII+
Ly6Chi
monocytes
(Gr-1+)a
MHCII+
14.1%
13.0%
MHCIIlo macrophages
Ly6Cneg-int F4/80+ MHCIIlo
21.1%
3.1%
MHCIIhi macrophages
Ly6Cneg-int F4/80+ MHCIIhi
15.8%
4.9%
CD11blo dendritic cells (DCs)
Ly6Cneg-int F4/80− MHCIIhi CD11chi CD11blo (CD103+)a
2.9%
0.4%
CD11bhi
Ly6Cneg-int
DCs
F4/80−
MHCIIhi
CD11chi
CD11bhi
(CD103−)a
2.3%
0.8%
Eosinophils
Ly6Cneg-int F4/80lo SSChi
2.9%
N.D.
Natural killer cells
Ly6Cneg-int F4/80− CD11b+ CD11c+ (indirect)a
9.6%
7.6%
T cells
Ly6Cneg-int F4/80− CD11b− CD11c− (indirect)a
5.8%
6.4%
89.5%
82.1%
Total identified N.D., not detectable. aSee Figure 1 legend for further explanation.
of Ly6C and from eosinophils based on the fact that eosinophils are uniquely FSClo SSChi. Importantly, F4/80 should be placed on one of the brighter fluorescent channels (i.e., PE, PE-Cy7, or APC) because overall expression of this marker is relatively low even on macrophages.
Monocytes Blood monocytes that have newly emerged from the bone marrow have a Ly6Chi surface phenotype and are thought to return to bone marrow to become Ly6Clo monocytes.8 Ly6Chi monocytes are abundant in implantation sites up until ∼gd 10.5, but extravasate rapidly only in the myometrium, where they differentiate into tissue macrophages. In contrast, they primarily remain intravascular in the decidua, where they show moderately downregulated Ly6C expression by gd 9.5. This downregulation can complicate their discrimination from macrophages. Like macrophages, Ly6Chi monocytes can be divided into two subsets based upon MHCII expression.
Neutrophils Neutrophils are readily detected in any mouse tissue based upon their high expression of Ly6G. They can also be identified with Gr-1 antibodies, which bind strongly to Ly6G and weakly to Ly6C; neutrophils thus appear Gr-1hi and Ly6Chi monocytes appear Gr-1lo. Importantly, due to the high expression of Gr-1 and Ly6C on neutrophils and monocytes, respectively, either of these markers can be placed on Alexa Fluor 700, the dimmest of the available fluorescent channels (Table 1). Although neutrophils constitute a major population in the decidua, as with monocytes,
many of the cells are intravascular and are bound to endothelial cells within the vascular zone.9
Dendritic Cells All DCs highly express MHCII and are differentiated from other tissue-resident APCs, such as macrophages and B cells, by their high expression of CD11c (B Cells are CD11c−) and low-to-absent expression level of F4/80 (macrophages are F4/80+). The pregnant mouse uterus harbors two subsets of DCs that differ based upon their expression of CD103 and CD11b.10
Eosinophils Eosinophils (Figure 1) are abundant in the murine nonpregnant uterus during estrus, but during pregnancy are only present within the myometrium (Table 2). They express intermediate levels of F4/80 and can be differentiated from other myeloid cells based on their uniquely high SSC properties.
Natural Killer Cells Using the staining panel optimized for myeloid cell types (Table 1), uNK cells can be indirectly identified as CD45+ cells that fail to express Ly6C, MHCII, or F4/80, but express both CD11b and CD11c.11 More directly, uNK cells are identified as CD3−CD122+.3 They can then be divided into subsets through the use of DX5 antibodies (or NK1.1 antibodies in certain mouse strains, namely C57BL/6, FVB/N, NZB, NZW, and SJL) and fluorescently labeled DBA lectin (see Chapter 19).
T Cells Using the staining panel optimized for myeloid cell types (Table 1), T cells in the pregnant mouse uterus
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can be indirectly identified as CD45+ cells that fail to express any of the other markers. More directly, they are identified using CD3 antibodies, and can be further subsetted using antibodies to TCRβ and TCRγ/δ (to differentiate αβ and γδ T cells), CD4 and CD8 to distinguish αβ T cell subsets, and by intracellular staining for select cytokines and FOXP3 to distinguish different CD4 T cell subsets. NKT cells are present at low numbers and mostly can be identified as CD3+ DX5+ or, in the appropriate mouse strains, as CD3+ NK1.1+ (see Section Natural Killer Cells).12
B Cells B cells are rare in nonpregnant and pregnant uteri under noninflammatory conditions.13 In principle, they can be identified as Ig+ MHCII+ CD19+.
Technical Limitations Although flow cytometry offers a highly sensitive and specific means to visualize all leukocyte subsets within the pregnant mouse uterus, it cannot provide information regarding the position of these cells in situ aside from a basic discrimination between the myometrium and decidua. Such information requires tissue immunostaining, as described in Chapter 49. Furthermore, analysis of a given uterine tissue layer will not be able to distinguish intravascular from extravascular leukocytes unless a specific technique (Section Determination of Uterine Leukocyte Blood/Tissue Partitioning) for this discrimination is employed.
the problem of placental contamination of decidual tissue has already been discussed, it should also be noted that the placenta contains fetal leukocytes, many of which are autofluorescent and can be gated away if one fluorescent channel is left open (Table 1). Finally, the availability of a three-laser (10-color) flow cytometer is not always guaranteed. We recommend the use of 10 colors for the identification of leukocyte populations when possible, but 6-color or 3-color formats can be used for more basic analyses.
References 1. Tagliani E, Shi C, Nancy P, Tay CS, Pamer EG, Erlebacher A. Coordinate regulation of tissue macrophage and dendritic cell population dynamics by CSF-1. J Exp Med August 29, 2011;208(9):1901–16. 2. Movahedi K, Laoui D, Gysemans C, Baeten M, Stange G, Van den Bossche J, et al. Different tumor microenvironments contain functionally distinct subsets of macrophages derived from Ly6C(high) monocytes. Cancer Res July 15, 2010;70(14):5728–39. 3. Yadi H, Burke S, Madeja Z, Hemberger M, Moffett A, Colucci F. Unique receptor repertoire in mouse uterine NK cells. J Immunol November 1, 2008;181(9):6140–7. 4. Fridman WH, Galon J, Pages F, Tartour E, Sautes-Fridman C, Kroemer G. Prognostic and predictive impact of intra- and peritumoral immune infiltrates. Cancer Res September 1, 2011;71(17):5601–5. 5. Kruse A, Merchant MJ, Hallmann R, Butcher EC. Evidence of specialized leukocyte-vascular homing interactions at the maternal/fetal interface. Eur J Immunol April 1999;29(4):1116–26. 6. Welsh AO, Enders AC. Light and electron microscopic examination of the mature decidual cells of the rat with emphasis on the antimesometrial decidua and its degeneration. Am J Anat January 1985;172(1):1–29. 7. Nancy P, Tagliani E, Tay CS, Asp P, Levy DE, Erlebacher A. Chemokine gene silencing in decidual stromal cells limits T cell access to the maternal-fetal interface. Science June 8, 2012;336(6086):1317–21. 8. Geissmann F, Manz MG, Jung S, Sieweke MH, Merad M, Ley K. evelopment of monocytes, macrophages, and dendritic cells. Science D February 5, 2010;327(5966):656–61.
Importantly, different cell subsets have varying capacities to survive enzymatic tissue digestion and the shear stress that accompanies cell acquisition. In particular, uNK cells, epithelial cells, and stromal cells are large and fragile; therefore, they are more susceptible to cell death. These subsets might consequently be underrepresented in FACS analyses. Certain cell preparation protocols have been developed to better preserve NK cells.3
9. Karsten CM, Kruse A. The role of vascular addressins in implantations sites during successful and failing mouse pregnancies. Immunol Invest 2008;37(5):449–66.
Another issue that can arise starting at gd 10.5 is placental contamination of decidual tissue (see Section General Leukocyte Analysis; Step 3). Although
13. Cha HR, Ko HJ, Kim ED, Chang SY, Seo SU, Cuburu N, et al. Mucosa-associated epithelial chemokine/CCL28 expression in the uterus attracts CCR10+ IgA plasma cells following mucosal vaccination via estrogen control. J Immunol September 15, 2011;187(6):3044–52.
10. Collins MK, Tay CS, Erlebacher A. Dendritic cell entrapment within the pregnant uterus inhibits immune surveillance of the maternal/fetal interface in mice. J Clin Invest July 2009;119(7):2062–73. 11. Mallidi TV, Craig LE, Schloemann SR, Riley JK. Murine endometrial and decidual NK1.1+ natural killer cells display a B220+CD11c+ cell surface phenotype. Biol Reprod August 2009;81(2):310–8. 12. Boyson JE, Aktan I, Barkhuff DA, Chant A. NKT cells at the maternal-fetal interface. Immunol Invest 2008;37(5):565–82.