Zombie red fixable

For flow cytometeric analysis, cells were washed with FACS buffer (1 × PBS, 2.5% FBS), resuspended in 100 μl FACS buffer, incubated with an Fc blocking reagent (BD PharMingen) and stained at 4^∘C with antibodies to various cell surface antigens. Viability was assessed with DAPI (Invitrogen) or Zombie red fixable (BioLegend). Cells were washed 1× with FACS buffer, and resuspended in 200 μl FACS buffer. Flow cytometric analysis of CFSE or Annexin V stainings was performed according to manufacturer’s instructions (CellTrace CFSE Cell Proliferation Kit, Invitrogen and FITC Annexin V Apoptosis Detection Kit, BD Biosciences. Briefly, purified naive B cells were stained with 5 μM CFSE and cultured for 96 h in LPS plus IL-4 and CFSE dilution was monitored by flow cytometer. GFP⁺ cells or bone marrow B cell populations were sorted in a FACSAria cell sorter (BD Biosciences). Flow cytometry was performed using an LSR-II flow cytometer (BD Biosciences), and data analyzed using FlowJo software (version 9.9).