Marilen Benner

CHAPTER 7 198 Lymphocyte subset analysis For flow cytometric analysis of lymphocytes, single cells suspensions were prepared from isolated placental tissue, spleens, mesenteric lymph nodes (MLN) and inguinal lymph nodes (ILN). ILN and MLN single cell suspensions were obtained by crushing the tissue through 70 µm cell strainers on ice. The strainers were washed with RPMI 1640 medium, after which the cells were counted, resuspended in PBS and kept on ice until further processing. Splenocytes were similarly isolated, but red blood cells were lysed prior to counting the cells using lysis buffer (8.3 g NH4Cl, 1 g KHC3O, and 37.2 mg EDTA dissolved in 1 L demi water and filter sterilized). Placental tissues were cut into small pieces and incubated with Accutase (Stempro, GIBCO Life Technologies, Waltham, USA) for 35 min at 37°C under slight agitation. Hereafter, red blood cells were lysed as described for splenocytes, and placental cells were washed, counted, resuspended in PBS and kept on ice until further processing. Prior to staining cells for flow cytometric analysis, they were washed in PBS and 50µl of cell suspension (4.10 6 cells/ml) was incubated with a fixable viability dye eFluor® 780 (eBiosciences, Thermo Fisher Scientific, San Diego, CA, USA) for 30 min at 4°C. After washing, cells were incubated with anti-mouse CD16/CD32 (1:100 dilution in PBS/1% BSA; Mouse BD Fc Block, BD Pharmingen, San Jose, CA, USA) to block non-specific binding sites. For flow cytometric analysis of surface marker expression, cells were incubated at room temperature for 1 h in the dark with corresponding antibody-cocktails, washed with PBS/1% BSA and fixed in 1 % paraformaldehyde-solution until flow cytometric analysis. For the analysis of intracellular markers, cells were first stained for extracellular markers, washed with PBS/1% BSA and incubated overnight in Fix/Perm buffer (eBiosciences). The following day, cells were washed with permeabilization buffer (eBioscience), and incubated with anti-mouse CD16/CD32 for 15 min at 4°C in the dark. Next, the cells were stained for intracellular markers for 30 min at 4°C in the dark, washed in PBS/1% BSA and immediately used for flow cytometric analysis. The following fluorochrome-conjugated monoclonal antibodies were used: CD4-PerCP-Cy5.5 (eBioscience), CD69-APC (eBioscience), CXCR3-PE (eBioscience), T1ST2-FITC (MD Biosciences, St. Paul, MN, USA); CD11b- PerCP-Cy5.5 (eBioscience), NK1.1-APC (eBioscience), CD49b-FITC (eBioscience), CD94-PE (eBioscience); CD4- Brilliant Violet 510, CCR6-PE (BioLegend, San Diego, CA, United States), CD25-PerCP-Cy5.5, (eBiosciences), CD196 (CCR6)-PE (BioLegend), CD127-PE-Vio770 REA (Miltenyi Biotec, Bergisch Gladbach, Germany), Neuropilin-eFluor450 (eBioscience), Ror γ T-Alexafluor 647 (BD Pharmingen, San Jose, CA, USA), CD1d-PerCP-Cy5.5 (BioLegend), CD5-Alexa Fluow 647 (BioLegend), CD19-PE-Cy7 (BD), CD21/CD35-FITC (BD), CD23-PE (BD), CD24-Brilliant Violet 510 (BD), Tim-1-Brilliant Violet 421 (BD), Viability-APC-Cy7 (eBioscience). Results were collected with BD FACSCanto II flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA) and analyzed with FlowLogic software (Inivai Technologies, Mentone, VIC, Australia) and Kaluza software (v2.1, Beckman Coulter, Fullerton, CA, USA).