Single-cell suspensions were prepared from lymphocytes of thymus (T), bone marrow (BM), spleen (Spl), and lymph node (LN) from mice of the described genotypes and ~1 × 105 cells in 1× PBS were stained for 15 min at RT using fluorescence-conjugated antibodies and analyzed by flow cytometry60 (link). The following antibodies were used: FITC-Cd11b (1:200, M1/70, BD Pharmingen, 553310), APC-Ly-6G/Ly-6C (Gr-1) (1:200, RB6-8C5, Biolegend), PE-IgM (1:400, SouthernBiotech, 1020-09), PE-CD4 (1:200, GK1.5, BD Pharmingen, 553730), FITC-CD8a (1:200, 53-6.7, Biolegend), PE-Cyanine5-CD3e (1:200, 145-2C11, Invitrogen, 15-0031-63).
For CSR assay, single-cell suspensions of spleen cells were sorted with CD43 magnetic beads (MACS, Miltenyi), and CD43- B cells were cultured at a density of 5 × 105 cells per ml in RPMI medium supplemented with 15% FBS and 25 ng ml−1 of IL-4 (R&D) and anti-CD40 (BD Biosciences). Cultured cells were maintained daily at a density of 1 × 106 cells per ml. Cells were collected every day up to day 4.5 and were stained with FITC-conjugated IgG1 (1:200, A85-1, BD Pharmingen, 553443) and PE-Cyanine5-conjugated B220 (1:200, RA3-6B2, BD Pharmingen, 553091). Flow cytometry was performed on a FACSCalibur flow cytometer (BD Biosciences) and data were processed using FlowJo software package.
For CSR assay, single-cell suspensions of spleen cells were sorted with CD43 magnetic beads (MACS, Miltenyi), and CD43- B cells were cultured at a density of 5 × 105 cells per ml in RPMI medium supplemented with 15% FBS and 25 ng ml−1 of IL-4 (R&D) and anti-CD40 (BD Biosciences). Cultured cells were maintained daily at a density of 1 × 106 cells per ml. Cells were collected every day up to day 4.5 and were stained with FITC-conjugated IgG1 (1:200, A85-1, BD Pharmingen, 553443) and PE-Cyanine5-conjugated B220 (1:200, RA3-6B2, BD Pharmingen, 553091). Flow cytometry was performed on a FACSCalibur flow cytometer (BD Biosciences) and data were processed using FlowJo software package.
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