Flow cytometry was performed as previously described [9 (link)]. Spleen and cervical lymph nodes (cLNs) were mechanically dissociated and treated with RBC lysis buffer. Cells were incubated with Fc block (Cd16/32, clone 2.4G2, BD Biosciences) and were treated with the following antibodies as indicated: B220 (clone RA3-6B2, BD Biosciences), CD23 (clone B3B4, BioLegend), CD21/35 (clone 7G6, BD Biosciences), CD4 (clone GK1.5, BD Biosciences), CD69 (clone H1.2F3, BioLegend), CD86 (Clone GL1, BD Biosciences), CD11c (clone HL3, BD Biosciences), CD11b (clone M1/70, BD Biosciences), and CD138 (Clone 281-2, BD Biosciences). Data were acquired using a Fortessa (BD Biosciences) and analyzed using FlowJo software (BD Biosciences).
Sort-purification was performed using a FACSAria (BD Biosciences). Splenocytes were incubated with antibodies directed against B220, CD4, CD11b and CD11c as described above. B cells (B220+), T cells (CD4+) or monocytes (CD11b+, CD11c+, and CD11b/CD11c double positive) (1 × 105 cells) were sorted directly into lysis buffer for RNA isolation (RNeasy Plus Mini Kit, Qiagen). For stimulation experiments, B220+ cells were sorted into media and cultured overnight with either culture media alone or culture medium containing either LPS or anti-IgM/IgG + IL-4, as described above.
Sort-purification was performed using a FACSAria (BD Biosciences). Splenocytes were incubated with antibodies directed against B220, CD4, CD11b and CD11c as described above. B cells (B220+), T cells (CD4+) or monocytes (CD11b+, CD11c+, and CD11b/CD11c double positive) (1 × 105 cells) were sorted directly into lysis buffer for RNA isolation (RNeasy Plus Mini Kit, Qiagen). For stimulation experiments, B220+ cells were sorted into media and cultured overnight with either culture media alone or culture medium containing either LPS or anti-IgM/IgG + IL-4, as described above.