10d after immunizing groups of control Cγ1-Cre+/−, DAF-TMCγ1 and ΔC3ar1/C5ar1Cγ1 mice with SRBC, animals were injected with anti-CD40 (150ug) and/or anti-IgM F(ab’)2 (100ug), or goat IgG2a (all from BioXcell) in 200ul PBS i.v. Spleens were harvested at 4h for surface staining followed by fixation, permeabilization and intracellular staining. In other studies, 20×106 B6 WT or C3aR1−/−C5aR1−/− B cells were transferred into μMT recipients and 24 h later immunized the recipients with SRBC. On d10, animals were injected with anti-CD40 (150ug) and/or anti-IgM F(ab’)2 (100ug), or control goat IgG2a and the cells analyzed by flow cytometry. Serum IgM was quantified with an IgM Mouse Uncoated ELISA Kit (Invitrogen).
For in vitro studies, 10d after sequential SRBC immunization, isolated spleen cells were enriched for B cells (B cell Magnisort kit, ThermoFisher), rested for 20min at 37C, 5% CO2 in HL-media and subsequently stimulated with 10ug/ml anti-CD40, 10ug/ml anti-IgM, 0.5ug/ml murine C3a and or C5a for 20’ at 37C, 5% CO2. After washing, cells were stained and analyzed cells by flow cytometry.
For in vitro studies, 10d after sequential SRBC immunization, isolated spleen cells were enriched for B cells (B cell Magnisort kit, ThermoFisher), rested for 20min at 37C, 5% CO2 in HL-media and subsequently stimulated with 10ug/ml anti-CD40, 10ug/ml anti-IgM, 0.5ug/ml murine C3a and or C5a for 20’ at 37C, 5% CO2. After washing, cells were stained and analyzed cells by flow cytometry.