Cd45r b220 apc

To quantify B cell populations in spleen and bone marrow single cell suspensions (10⁶ cells/reaction) were prepared and surface stained in FACS buffer (phosphate-buffered saline (PBS) containing 1% Fetal Bovine Serum (FBS) for 30 minutes at 4°C, and washed once in FACS buffer. B cells were stained using CD45R (B220)-APC or IgD-FITC (eBioscience Inc. (San Diego, CA) and gates established using isotype and fluorochrome-matched controls. Samples were acquired on an Accuri flow cytometer (BD Immunocytometry Systems).

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.