Cd69 pe clone l78

Freshly obtained peripheral mononuclear cells (PBMCs) were isolated after performing Ficoll density gradient centrifugation. Naive B cells were isolated from PBMCs using the MACS naive B cell isolation kit II in an Automacs (Miltenyi Biotech). Naive B cell enrichment was assessed using CD19 PerCP-Cy5.5, CD27 FITC, CD3 APC (BD Biosciences) and IgD PE (Southern Biotech). We resuspended 1 × 10 5 purified naive B cells in 200 µl RPMI-1640 supplemented medium (10% fetal calf serum, 100 U/ml penicillium, 100 µg/ml streptomycin and 2 mM glutamine). Cells were activated for 4 days with 2 μg/ml of MegaCD40L soluble human recombinant (Enzo Life Sciences, Farmingdale, NY, USA) and 100 ng/ml of human recombinant IL-21 (Gibco, Life Technologies, Carlsbad, CA, USA); unstimulated samples were performed in parallel. Cell cultures were maintained for 4 days at 37°C in a 5% CO 2 atmosphere. Cell activation was measured with the monoclonal antibody combinations CD19 PerCP-Cy5.5, CD27 FITC, CD69 PE (clone L78) and CD86 APC (clone 2331 Fun-1) (BD Biosciences). Responsiveness to stimulation was calculated as the fold increase of MFI in CD69 and CD86 in stimulated cells to the same markers in the unstimulated cells (fold change = MFI-stimulated/MFIunstimulated). The analysis was performed by flow cytometry using FACS Canto II (BD Biosciences) and the FlowJo Software (LLC).