Cd45r b220 apc

For the in vitro stimulation of splenic B cells and bone marrow cells, we used goat anti-mouse IgM (Southern Biotechnology, 1020-01) antibody. For BCR internalization experiments, we used rat anti-mouse IgM-FITC (BD Pharmingen, R6-60-2) and goat anti-mouse IgM-pHrodo (Southern Biotechnology, 1020-01) antibodies. Apoptosis assays were performed with annexin V-FITC and the corresponding binding buffer (eBioscience) with eFluor780 and eFluor450 (eBioscience). For FACS analysis, the cells were further stained with CD45R/B220-APC (BD Pharmingen, clone RA3-6B2) antibody. For co-IP, we used anti-HAX1 (BD Transduction Lab) and anti-human IgE-HRP (KPL) antibodies.

Single cell suspensions of PP cells were isolated as described55 (link). For flow cytometry, isolated cells were transferred to a 96 round-bottom well plate and subjected to centrifugation at 1000 × g for 5 min. Fc receptors were blocked for 15 min with supernatants from the ATCC 2.4.G2 cell line before the addition of the primary antibody cocktail (20 ug/ml). Cells were incubated on ice for 30 min with continuous rocking and then washed and fixed in PHEM buffer containing 1% paraformaldehyde and subjected to flow cytometry using a FACS Calibur. Results were analyzed using Cell Quest Pro software version 5.2. For imaging flow cytometry was performed using the Image Stream (Amnis, Seattle WA) equipped with a 480–560 laser. Cells (1 × 10⁶) were prepared in 50 μl of PHEM buffer containing 1% paraformaldehyde. Anti-mouse Abs CD45R/B220-APC, CD3-FITC, CD4-PE, CD8-PE were purchased from BD Biosciences (Franklin Lakes, NJ); CD45R/B220-APC was from eBioscience and CD19-PercP was from BioLegend.