Flow Cytometry of Mouse Splenic B Cells

Spleens were collected and pressed through 70-μm cell strainers with complete medium (RPMI 1640, 10% fetal bovine serum, 1 mM sodium pyruvate, 1% 100 MEM non-essential amino acids, 10 mM HEPES, 55 mM 2-mercaptoethanol, 2 mM L-glutamine, 100 U/ml penicillin-streptomycin, all from Life Technologies, Grand Island, NY). Red blood cells (RBCs) were excluded using previously published methods (30 (link)). Single-cell suspensions were stimulated for 24 hours with 10 μg/ml LPS, followed by 5 hours of 50 ng/ml PMA and 500 ng/ml ionomycin, blocked for 10 minutes on ice with the Fc receptor block anti-CD16/32 (eBioscience), then stained for 15 minutes in the dark with fluorochrome-conjugated antibodies and analyzed with a BD FACS Aria II flow cytometer (BD Biosciences, San Jose, CA). Foxp3 Fixation/Permeabilization kit (eBioscience) was used for intracellular staining. To exclude dead cells, a Zombie Aqua fixable viability kit (Biolegend) was used. The following monoclonal anti-mouse antibodies were used in this study: AF700 or APC conjugated anti-CD19 diluted 1:800, PE-Cy7 conjugated anti-CD23 diluted 1:60, APC-Cy7 conjugated anti-CD21 diluted 1:80, FITC conjugated anti-CD24 diluted 1:200, PE conjugated anti-IL-10 diluted 1:80, BV711 conjugated anti-CD138 diluted 1:800, and APC conjugated anti-IgH (μ chain) diluted 1:800 (BioLegend).