B Cell Cultures and CellTrace Violet Staining

B cells were isolated from single-cell splenocyte suspensions using B cell isolation kits (Miltenyi Biotec, 130090862) and MACS Separation LS columns (Miltenyi Biotec, 130042401) following the manufacturer’s instructions. 10 million isolated B cells were incubated with CellTrace™ Violet (CTV; Thermo Fisher Scientific, {"type":"entrez-nucleotide","attrs":{"text":"C34557","term_id":"2370698","term_text":"C34557"}}C34557) at 1:1,000 dilution at 37°C in the dark for 20 min, centrifuged at 300 × g for 10 min, and the cell pellet was washed once with 10 mL of MACS buffer. CTV-stained B cells were cultured in the B cell culture media (RPMI 1640 supplemented with 5% (v/v) FBS, 50 µM β-mercaptoethanol, 100 U/mL Penicillin G and 100 µg/mL streptomycin sulfate) at a seeding density of 1 million cells/mL. Cells were stimulated with 15 µg/mL lipopolysaccharide (LPS; Jomar Life Research, tlrl-3pelps) or with 1/1,000 recombinant CD40L plus 1/100 conditioned mouse IL4 supernatant for 96 h, and applied to flow cytometry analysis (BD LSRFortessa). Division and proliferation indices were determined using the cell proliferation module of FlowJo 10.3.