Anti fcγ 3 2 receptor

The following antibodies and reagents were used at the indicated concentrations. Allophycocyanin (APC)-conjugated or phycoerythrin (PE)-conjugated anti-mouse CD45R/B220 (RA3-6B2; 2 µg/ml), fluorescein isothiocyanate (FITC)- or PE-conjugated anti-IgM (R6-60.2; 5 µg/ml), APC-anti-CD19 (1D3; 2 µg/ml), FITC-anti-IgD (11-26c.2a; 5 µg/ml), FITC-anti-CD21/CD35 (7G6; 10 µg/ml), biotinylated anti-heat stable antigen (HSA; M1/69; 0.1 µg/ml), FITC-anti-CD45.1 (A20; 2 µg/ml), PE-anti-CD38 (90; 0.7 µg/ml), PE-anti-IgG1 (A85-1; 1 µg/ml), PE-anti-CD138 (281-1; 2 µg/ml), PE-conjugated streptavidin (1 µg/ml) or PerCP-5.5cyanine-conjugated streptavidin (0.5 µg/ml) were from BD Bioscience. biotinylated anti-GL7 (GL7; 2 µg/ml) purchased from eBioscience. Alexa Fluor 647-labeled HEL was prepared with an Alexa Fluor647 antibody-labeling kit (Invitrogen). Before staining with the antibodies, cells were incubated for 10 min on ice with anti-Fcγ III/II receptor (2.4G2; 0.5 µg/ml; BD Bioscience) to block Fc receptors. In some experiments, mice were injected intraperitoneally with 300 µl of BrdU (Invitrogen) and sacrificed 5 h later. Splenocytes were then stained with APC BrdU flow kit (Becton-Dickinson). Flow cytometric analyses were done on FACS Calibur (BD Bioscience).