Anti mouse cd16 cd32 2.4g2

Spleens were isolated from C57BL/6 mice and were dissociated into single-cell suspensions by treatment with collagenase type II enzyme (Sigma-C6885), in order to facilitate separation from extracellular matrix and other unwanted tissue-derived material that might mask the ionization of low abundant peptides, and to promote detergent solubilization. Splenic B cells were evaluated for I-A^b expression by gating on the B220⁺ CD43^- CD11b^- population. The cells were blocked with 50 µg/ml anti-mouse CD16/CD32 (2.4G2, BioXCell, West Lebanon, NH) prior to staining. Cells were acquired on an LSR II flow cytometer (Becton Dickinson) and analyzed using FlowJo version 9.8.5 software (Tree Star, Ashland, OR). To isolate mature B cells from the splenocyte population, CD43^- and CD11b^- expressing cells were depleted using biotinylated anti-mouse CD43 and CD11b (BioLegend) in conjunction with the EasySep Mouse Streptavidin RapidSpheres Isolation Kit (Stem Cell Technologies, Cambridge, MA) according to the manufacturer’s instructions. Purity post-isolation was determined by FACS to be >90% for each sample, with an average purity of 94 ± 2.6%. Splenic B cells were isolated from 9 mice for each replicate sample.