Marilen Benner

CHAPTER 4 96 fluorochrome-conjugated monoclonal antibodies (moAbs) of interest for 20 min at RT in the dark. For Treg subset characterization and chemokine markers assessment a minimum of 500.000 mononuclear cells were used; 50.000 for intracellular cytokine assessment. For intracellular staining, fixation and permeabilization was carried out according to manufacturer’s instructions (eBioscience, San Diego, USA). IL-10 detection was preceded by incubation with Fixable Viability Dye-eFluor 780/Krome Orange (eBioscience) in PBS for 30min at 4 °C in the dark. Samples were measured using a 10-color Navios TM flow cytometer (Beckman Coulter, Fullerton, CA, USA). For manual gating, data were analyzed using Kaluza V2.1 (Beckman Coulter). Gates were set based on isotype controls and a fluorescent-minus-one strategy. Locations of gates were fixed except for CD3/CD56 based selection of NK cells as shown in Supplemental Figure S1. A detailed gating strategy for the various cell subsets and tissue types is shown in Figure S1B. CITRUS clustering For unsupervised analysis, manually gated lymphocytes (Figure 1) or CD3 negative lymphocytes (for B cell analysis in Figure 3) were extracted (plug in provided by Beckman Coulter) before further processing using the web-based analysis platform Cytobank (http://cytobank.org/) (61). Lymphocytes and CD19 + B cells, respectively, were chosen as input population. Files were assigned to the appropriate gestational-age group, i.e. menstrual blood, 1st trimester, 2 nd trimester and term. Unsupervised clustering of lymphocyte and B cell frequencies was performed using the CITRUS tool. Clustering was performed based on abundance with a minimum cluster size of 2% of total cells and a false discovery rate of 1%. For B cell clustering, samples of less than 200 B cell events were excluded from analysis (lowest amount of events included was 246). Clustering was analyzed with prediction analysis for microarrays (PAM), a predictive model to identify features determining gestational age-dependent variation. B cell stimulation assays 50.000 peripheral blood mononuclear cells or decidual lymphocytes were cultured in the presence or absence of CpG (5µg/ml) and CD40L (1µg/ml) stimulation, in 10% HPS culture media (RPMI 1640 medium supplemented with 1 mM pyruvate, 2 mM glutamax, 100 U/ml penicillin, and 100 μg/ml streptomycin) in a 96-well U-bottom plate for 24h at 37°C in a humidified 5% CO2 incubator. Phorbol-12-myristate-13-acetate (PMA, 50 ng/ml) and ionomycin (1 ug/ml) were added for the final 5h and brefeldin A (5µg/ml; Sigma-Aldrich, St. Louis, USA) for the final 2h of incubation. IL-10 expression of viable B cells was measured by flow cytometry as described. Immunohistochemistry Tissue samples were fixed in neutral buffered 4% formalin (Mallinckrodt Baker Inc, Deventer, The Netherlands) for 4h, transferred to 70% ethanol before preparation in a Tissue-Tek VIP tissue processor for embedding in paraffin. Slides were deparaffinized in xylene before rehydration, washing in tap water and boiling in Tris-EDTA buffer (pH 9, Klinipath). 6 μm sections were stained for either IHC or IF staining. For IHC staining, antibody binding was visualized by diaminobenzidine