B220 alexa fluor 647

For the CFSE proliferation assay, purified B cells were resuspended at 1 × 10⁷ cells/mL in RPMI medium, labeled with 5 μM CFSE for 10 minutes at 37°C, then cultured in RPMI medium with or without IL-4 and LPS for 72 hours. The CFSE fluorescence of unstimulated cells was taken as 100%, and the number of additional cell divisions was calculated according to reduced CFSE fluorescence as the cells divided. Number of cell divisions was calculated as log₂(median intensity of unstimulated cells / median intensity of indicated cells). For EdU incorporation, B cells were pulsed with 30 μM EdU for 30 minutes, and fixed with ice-cold methanol for 20 minutes. EdU was detected using the Click-iT EdU Alexa Fluor 647 imaging kit. Cells were stained with DAPI to measure DNA content. For flow cytometry analysis of splenocyte populations, mouse splenocytes were treated with ACK lysis buffer for 5 minutes, then blocked with anti-CD16/CD32 antibody for 10 minutes at room temperature and labeled with B220–Alexa Fluor 647 (557683, BD Biosciences) and CD43-PE (12-0431-82, Invitrogen) for 1 hour at 4°C. Cytometry was performed using a BD FACSCalibur or Cytek Aurora with analysis in FlowJo.