Anti ly6c fitc clone hk1.4

Flow cytometry analysis of splenic and peritoneal B cell subsets was performed as described previously 6 (link), 13 (link) using directly conjugated antibodies on single cell suspensions of freshly isolated spleens and peritoneal cells. Peripheral blood was diluted with PBS + 2% dextran (Sigma) for at least 30 minutes at 37°C to concentrate the RBCs at the bottom of the tube. The upper clear phase was collected, and cells were incubated with blocking anti-CD16/32 antibody (clone 93; eBiosciences). Peripheral monocytes and neutrophils were identified by staining with anti-CD11b APC (clone M1/70; eBiosciences), anti-Ly6C FITC (clone HK1.4; Biolegend) and anti-Ly6G-PE (clone 1A8; Biolegend). Resident peritoneal and splenic macrophages were stained with an anti-F4/80 antibody conjugated to APC (clone BM8; Biolegend). To quantify TACI expression, cells were stained with anti-TACI PE / APC and isotype control (anti-mouse IgD; clone 11-26c; eBiosciences) where indicated. T regulatory cells were quantified using the Treg detection kit (Miltenyi). Data were acquired on a FACS Calibur (BD) or LSRII Fortessa (BD) and were analyzed using Flow Jo software 7.6 (Treestar).