Primary cells were isolated from male 6–8 week-old C57Bl/6 mice (Charles River Laboratories). Peritoneal macrophages were harvested by peritoneal lavage 3 days after i.p. injection of 3 ml thioglycollate, overnight culture and adherence selection. BMDM were generated as described (Valledor et al., 2004). Splenic B cells were isolated by magnetic depletion of CD43- and CD11b–expressing cells (Miltenyi). B220+ pro-B cells were magnetically enriched from bone marrow of Rag1 knockout mice and expanded for 10 days as described (Sayegh et al., 2005) with slight modifications. E2A−/−and EBF−/− pre-pro-B cells were cultured as described previously (Ikawa et al., 2004). PU.1−/− and PUER cells were propagated and the PU.1-ER fusion protein was activated with 100 nM 4-hydroxy-tamoxifen as described (Walsh et al., 2002).
