Marilen Benner

CHAPTER 2 38 cells were washed twice with PBS. Menstrual blood was washed with PBS and passed through a 70 µm cell strainer (Falcon, Durham, USA) to remove clots and mucus. Granulocytes were depleted by use of a granulocyte depletion kit according to the manufacturer’s instructions (STEMCELL Technologies, Vancouver, Canada). After isolation, cells were washed twice with PBS containing 2% HPS. Decidua parietalis was collected as described previously (30). Briefly, after removing the amnion, the decidua parietalis was carefully scraped from the chorion. The obtained tissue was washed thoroughly in PBS before mincing with scissors. The resulting pulp was washed again until the supernatant became transparent. The tissue was enzymatically incubated with 1% collagenase I (Gibco Life Technologies, Waltham, USA) and 1% DNAse (Roche Diagnostics, Risch-Rotkreuz, Switzerland) in a water bath at 37 0 C while shaking for 60 minutes. After washing with RPMI medium, the suspension was passed through a 70 μm cell strainer (Greiner, Frickenhausen, Germany) and washed again with RPMI. Lymphocytes were obtained after density gradient centrifugation (Lymphoprep). Analysis was done immediately on fresh material to exclude the influence of cryopreservation on the expression of certain markers. For optimal analysis of the chemokine receptors CD183 (CXCR3), CD194 (CCR4), and CD196 (CCR6) on decidual cells, cells were put to rest at 37°C in a humidified 5% CO 2 incubator for 16 hours before staining for flow cytometry. Typically 93%, 95%, and 81% of respectively peripheral, menstrual, and decidual lymphocytes were viable cells (Supplementary Figure S1). Flow cytometry Samples were phenotypically analyzed using the 10-color NaviosTM flow cytometer (Beckman Coulter, Fullerton, CA, USA). Briefly, cells were washed twice with PBS + 0.2% bovine serum albumin (BSA; Sigma-Aldrich, St. Louis, USA) and labeled for 20 min at RT in the dark with the fluorochrome-conjugated mAbs of interest. Samples were washed twice with PBS + 0.2% BSA. For cell surface staining of B cells, monocytes/macrophages, T cells, Treg, NKT cells and NK cells, the following conjugated mAbs were used: CD3-PE/ECD/PB (Beckman Coulter; UCHT1), CD4-PC5.5/PB (Beckman Coulter; 13B8.2), CD4-AF700 (eBioscience, San Diego, USA; RPA-T4), CD8-APC-AF700/APC-AF750 (Beckman Coulter; B9.11), CD14-ECD (Beckman Coulter; RMO52), CD16-FITC (Beckman Coulter; 3G8), CD19-APC-AF750 (Beckman Coulter; J3-119), CD25-PC7/APC (BD Biosciences, New Jersey, USA; M-A251 and 2A3), CD45-KO (Beckman Coulter; J33), CD45RA-FITC/ECD (Beckman Coulter; ALB11 and 2H4LDH11LDB9), CD45RO-ECD (Beckman Coulter; UCHL1), CD56-APC (Beckman Coulter; N901), CD62L-FITC/ ECD (eBiosience and Beckman Coulter; DREG-56), CD69-PE (Beckman Coulter; TP1.55.3), CD103-FITC (eBioscience; B-Ly7), CD127-APC-AF700 (Beckman Coulter; R34.34), CD183- PC5.5 (CXCR3; Biolegend, San Diego, USA; G025H7), CD194-PC7 (CXCR4; BD Biosciences; 1G1), CD196-PE (CCR6; BD Biosciences; 11A9), CD197-BV421 (CCR7; BioLegend; G043H7), and Fixable Viability Dye-eFluor780 (eBioscience). For intracellular staining, samples were permeabilized and fixed according to manufacturer’s instructions (eBioscience). Cells were incubated with the conjugated mAbs of interest for 30 min at 4°C in the dark. The following