Mouse spleen cell suspensions in RPMI medium were filtered (70-micron), RBCs lysed (ACK buffer, Lonza) and re-filtered (40-micron) to produce single mononuclear cell suspensions in PBS plus 1%FCS. For primary murine and human cells as well as cell lines, surface and intracellular staining were performed using antibodies listed in the Resource Table. Analyses were performed on FACS Lyric™ or Canto™ II cytometers (BD Biosciences) and the results analyzed with FlowJo software version 10.6.2 (FlowJo LLC).
GCB cells from the spleens of SRBC immunized mice were sorted after enrichment of by negative selection, using the MagniSort Mouse B cells Enrichment Kit (ThermoFisher Scientific), with addition of anti-IgD-biotin antibody to deplete IgD+ naïve cells. GC B cell pools were sorted using GL7+FAS+ (Cγ1-cre+/− control mice) and GL7+FAS+ DAF+ (DAF-TMCγ1 mice) in a BD FACS Aria ™ III. DAF expression was used in the GL7+/FAS+ fraction to confirm transgene expression and successful recombination. In other analyses, cells in S phase were in vivo labeled for 2 h upon i.v. injection with 1 mg of 5-ethynyl-20-deoxyuridine (EdU)65 (link). Cells in S phase were gated as BrdU+ 7AADint.
GCB cells from the spleens of SRBC immunized mice were sorted after enrichment of by negative selection, using the MagniSort Mouse B cells Enrichment Kit (ThermoFisher Scientific), with addition of anti-IgD-biotin antibody to deplete IgD+ naïve cells. GC B cell pools were sorted using GL7+FAS+ (Cγ1-cre+/− control mice) and GL7+FAS+ DAF+ (DAF-TMCγ1 mice) in a BD FACS Aria ™ III. DAF expression was used in the GL7+/FAS+ fraction to confirm transgene expression and successful recombination. In other analyses, cells in S phase were in vivo labeled for 2 h upon i.v. injection with 1 mg of 5-ethynyl-20-deoxyuridine (EdU)65 (link). Cells in S phase were gated as BrdU+ 7AADint.
