Fitc conjugated cd25

CBMCs and peripheral whole blood were stained for 30 min at room temperature in the dark with the following monoclonal antibodies: PE Cy7-conjugated CD4, APC-Cy7-conjugated CD8, FITC-conjugated CD25, PerCP-conjugated CD62L, APC-conjugated CXCR3, PE-conjugated CCR4, APC-conjugated CD69, PerCP-conjugated HLA-DR, FITC-conjugated CD45RA, PE-conjugated CD45RO in separate tubes, respectively (all from BD Biosciences). After lysing red blood cells and washing, CBMCs and PBMCs were analyzed on a BD FACSAria flow cytometer (BD Biosciences) equipped with 488 nm and 633 nm excitation lasers. Data were processed using the FACSDiVa software. Figure 1 demonstrates the gating strategy applied.Fig. 1

Gating strategy of flow cytometry measurements. Example of a representative sample. FSC – forward scatter, SSC – side scatter

Th1 cells were defined as CD4+ CXCR3+, while Th2 cells were defined as CD4+ CCR4+. Naïve T cells were defined as CD4+ CD45RA+, while memory T cells were defined as CD4+ CD45RO+.

9 ml of peripheral venous blood was collected from each woman in lithium heparin-treated tubes (BD Vacutainer, BD Biosciences, San Jose, CA). Peripheral blood mononuclear cells (PBMCs) were isolated by standard density gradient centrifugation (Ficoll Paque, Amersham Biosciences AB, Uppsala, Sweden; 25 min, 400 g, room temperature). Cells were kept at −80°C in Fetal Bovine Serum containing 10% DMSO until analysis. After thawing, cells were washed twice in phosphate-buffered saline.
PBMCs were incubated for 30 min at room temperature in the dark with the following antibodies: APC-Cy7-conjugated CD4, PerCP-conjugated HLA-DR, APC-conjugated CD62E, PE-Cy7-conjugated CD62L, PE-conjugated CD62P, FITC-conjugated CD25, PE-Cy7-conjugated CD11c, PE-conjugated CD122, and APC-conjugated CD69 (BD Biosciences). After washing, fluorescent data were registered on a BD FACSAria flow cytometer (BD Biosciences). Data were processed using the FACSDiVa software. 200000 cells per sample were recorded.

Single-cell suspensions were stained in fluorescence-activated cell sorting (FACS) buffer at 4 °C for 30 min. We analyzed samples using an LSRII flow cytometer (BD Pharmingen, San Diego, CA). The following antibodies against mouse antigens were purchased from BD Pharmingen (San Diego, CA): BV605-conjugated anti-CD11b, FITC-conjugated anti-CD11c, BV450-conjugated anti-CD4, FITC-conjugated CD25, and PE-conjugated anti-Foxp3.