The rather large nucleus and very narrow cytoplasm of Treg cells makes it difficult to discriminate Small molecule library screening clearly the brown from the red staining. After trying different combinations for the cell surface and nuclear staining, we settled for the combination of brown (DAB) for surface staining and red (AEC) for nuclear staining which gave the best color discrimination. Each decidual tissue sample was macroscopically separated from the chorionic villi and washed thoroughly in Hank’s solution to get rid of debris and contaminating

blood. Decidual mononuclear cells (DMC) were isolated as previously described.36 Briefly, decidual tissue was cut into small pieces and filtered through a 60-μm stainless steel mesh to make

single cell suspension. The resultant suspension was subjected to Percoll (Pharmacia) density gradient centrifugation. Belnacasan cost The interface between 40 and 80% Percoll, containing mononuclear cells and epithelial cells, was collected. Contaminating epithelial cells were removed by incubation with mAb BerEP4 and goat anti-mouse magnetic beads (Dynabeads M-450; Dynal, Oslo, Norway), followed by magnet treatment. Peripheral blood mononuclear cells (PBMC) from pregnant and non-pregnant donors were isolated by Lymphoprep (Nycomed, Norway) gradient centrifugation according to the manufacturer’s instructions. Immunoflorescence staining and three color FACS analyses were performed in 9 consecutive paired DMC and PBMC samples from pregnant women and 9 PBMC from non-pregnant women. For immunofluorescence staining, we used Human Regulatory T cell Staining kit (eBioscience) according to the manufacturer’s instructions. In brief, one million (1 × 106) DMC or PBMC per tube were incubated with CD4-FITC/CD25-APC cocktail for 30 min in dark at 4°C. After

washing with cold FC staining buffer, the cells were permeabilized with Fix/Perm buffer for 30 min in dark at 4°C, and non-specific binding was blocked by incubation with 2% normal rat serum for 15 min. Then, Fossariinae the cells were incubated with Foxp3-PE Ab for 30 min, in dark, at 4°C. Control stains were included – positive control staining with CD45-FITC/CD14-PE (Dako) and isotype control staining with mouse IgG2a. In addition, PBMC and DMC suspensions were double stained with Foxp3 and CD56 (MY31), CD8-FITC (DK25), pan-γδ-FITC (5A6.E9) and Vδ1-FITC (TS8.2) mAbs, and goat anti-mouse IgG Fab-FITC (F0479). Percentages of Foxp3-positive cells were calculated within the CD4+ CD25−, CD4+ CD25+, and CD4+ CD25++ cell fractions. Data were acquired on FACS Calibur instrument (Becton Dickinson, San Jose, CA, USA) and analyzed using cellquest software (BD).