Marilen Benner

B CELLS CONTRIBUTE TO DECIDUAL IMMUNITY 95 4 and second trimester tissue was transported to the lab at room temperature and processed immediately upon arrival. Cell isolation Flow cytometry staining was performed on either whole blood or, for Treg staining, on isolated mononuclear cell populations. Peripheral blood mononuclear cells were isolated from whole blood based on density gradient separation (1.077 ± 0.001 g/ml, 290 ± 15mOsm, Lymphoprep™, Axis-Shield, Oslo, Norway). Menstrual blood mononuclear cells were isolated following an established protocol (58, 59). This protocol typically results in cell viability of ~96%. In brief, menstrual blood was washed with PBS (Braun, Melsungen, Germany) and filtered (70μm cell strainer Falcon®, Corning Inc., NY, USA) to remove clots or excessmucus. A granulocyte depletion cocktail RosetteSep™ (Stemcell technologies Inc, Vancouver, Canada) was used according to manufacturer’s instructions. After sterile density gradient centrifugation (Lymphoprep™), menstrual blood mononuclear cells (MMC) were collected. First and 2 nd trimester decidual leucocytes were isolated from maternal mucosal tissue that was carefully selected during visual assessment of the individual tissue pieces. Villous tissue, blood clots, glands and areas of blood infiltration were discarded. At these early stages of gestation, the selected pieces of membrane do not contain fused fetal membranes yet. Tissue pieces were minced mechanically using scissors and washed in PBS until the supernatant became transparent. For cells used for phenotyping, tissue was transferred to c-tubes (gentleMACS system) for additional mechanic processing using a MACS dissociator (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) prior to incubation with Accutase (Stempro, Gibco Life Technologies, Waltham, USA) in a 1:2 tissue to enzyme ratio; a gentle dissociation method to preserve marker expression (60). For B cell assays, washed tissue was digested using 0.2% collagenase (Gibco Life Technologies, Waltham, USA) and 0.04% DNAse (Roche Diagnostics, Risch-Rotkreuz, Switzerland). Tissue digestion was performed in a shaking water bath at 37 °C for 45 minutes. After digestion, the solution was filtered through 100μm, 70μm and 40μm cell strainers consecutively. Lymphocytes were obtained through density gradient centrifugation by diluting the washed filtrate in 20 mL 1.023 g/ml Percoll (GE Healthcare, Little Chalfont, UK) to be layered on gradient of 10 mL 1.080 g/ml and 15ml 1.053 g/ml Percoll (centrifugation for 25min at 2000rpm). Large cells accumulating at the 1.053 – 1.023 g/ml interface were discarded and decidual leucocytes were isolated from the 1.080 – 1.053 g/ml interface. Leucocytes were washed twice in RPMI before further use. For term decidua, decidua parietalis was separated from the two fetal layers. The amnion was removed prior to scraping decidua parietalis from the chorionic membrane. After this, the isolation procedure was identical to that of 1 st and 2 nd trimester. Flow cytometry staining and analysis For whole blood staining, 25ml red blood cell lysis buffer [NH 4 CL + KHCO 3 / Na 4 EDTA (Merck, Darmstadt, Germany) diluted in H 2 O (Versol, Lyon, France)] was added to 1ml of peripheral blood for 10 min, and washed 3times with PBS. A minimum of 200.000 cells was used for staining with