B220 alexa700

To identify the different B cell populations, two stains were performed in splenocytes from 4 to 5.5 month old mice (2 females and 1 male wt, and 4 females Kmt2d^−/−). First, to identify transitional, follicular and marginal zone populations, cells were stained with CD21-FITC (Biolegend, clone 7E9, #123407), CD5-PE (eBioscience, clone 53–7.3 #12-0051-81), CD23-PECY7 (Biolegend, clone B3B4 #101613), IgM-APC (Biolegend, clone RMM-1 #406509), and B220-Alexa700 (Biolegend, clone RA3 #103232). To identify intermediate plasma cells/plasmablast (IPC), plasma cells (PC) and germinal center populations, cells were stained with GL7-FITC (Biolegend, clone GL7 #144003), CD138-PE (Biolegend, clone 281-2 #142503), CD95-APC (eBioscience, clone 15A7 #17-0951-80), B220-Alexa700 (Biolegend, clone RA3 #103232). To determine the percentages of cell populations, values were normalized by % B220⁺ single live cells (single cells, 7-AAD negative. B220⁺). 7-AAD (Life Technologies) was used to identify dead cells. Data acquisition was performed in a BD LSR II Flow Cytometer (BD Biosciences) and analysis was performed with FlowJo software (Tree Star).

For class switch recombination to IgG1 resting splenic B cells were isolated from 2.5 to 5 months old Kmt2d^+/+ CD19-Cre- (wt, 2 females and 3 males) and Kmt2d^f/f CD19-Cre⁺ (Kmt2d^−/− 2 females and 3 males) mice by immunomagnetic depletion with anti-CD43 MicroBeads (anti-Ly48, Miltenyi Biotech), and cultured at 0.5×106 cells/ml with LPS (25 µg/ml; Sigma), IL-4 (5 ng/ml; Sigma) and RP105 (Anti-Mouse CD180; 0.5 µg/ml; BD Pharmingen) for 4 days. B cells were infected at 24 and 48 hours in culture with pMX-Cre-IRES-GFP as described³⁸ to enhance Kmt2df/f deletion. Class switching to IgG1 was measured at 96 hours in the GFP⁺ population (>90%) by flow cytometry using the following antibodies: IgG1-biotin (BD Pharmingen, clone A85-1 #553441) following streptavidin-Pacific Bule (Molecular Probes), B220-Alexa700 (Biolegend, clone RA3 #103232). Data acquisition was performed on the BD LSR II Flow Cytometer (BD Biosciences) equipped with CellQuest software (Becton Dickinson). Analysis was performed with FlowJo software (Tree Star).