Facs influx sorter

Bone marrow (BM) cells were collected by flushing femoral shafts with cold RPMI supplemented with 2% bovine serum albumin (BSA, US Biological, Swampscott, MA, USA) and 2 mM EDTA (IBI Scientific, USA). After depleting red blood cells using lysis buffer (Sigma-Aldrich, St. Louis, Missouri, USA), the cells were incubated with anti-B220 microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany), and the B-cells were isolated using positive selection with a magnetic activated cell-sorting (MACS) system (Miltenyi Biotec, Bergisch Gladbach, Germany). Single-cell suspensions of B220⁺ B-cells from BM were incubated with fluorescently labeled antibodies specific for CD43, B220, IgM, and CD23 in staining buffer (PBS with 0.5% BSA) for 20 minutes at 4°C, and cells were incubated with DAPI to select live cells (DAPI⁻). The cells were washed, and the immature B-cells were isolated according to the expression of the following surface markers: B220⁺, CD43⁻ CD23⁻, IgM⁺, and DAPI⁻. Cell sorting was performed using a FACS Influx Sorter (BD Biosciences). The purity of the sorted cells ranged from 95% to 98%.

Single-cell suspensions of B220+ B cells from BM were incubated with fluorescently labeled antibodies specific for CD43, B220, IgM, and CD23 in a staining buffer (PBS with 0.5% BSA) for 20 min at 4 °C. Further, the cells were incubated with DAPI to select living cells (DAPI−) and washed, then pro-B (B220+CD23-CD43+IgM–), pre-B (B220+CD23–CD43–IgM–), and immature B cells (B220+CD23–CD43–IgM+) were isolated [11 (link)]. Cell sorting was performed using a FACS Influx Sorter (BD Biosciences). The purity of sorted cells ranged from 95% to 98%.

Eighteen-week-old mice were euthanized, and spleen cells were collected with cold RPMI supplemented with 2% FBS and 2 mM EDTA (IBI Scientific, USA). Red blood cells were depleted with lysis buffer (Sigma-Aldrich, USA) and incubated with anti-CD4 MicroBeads (for T CD4 cells, Miltenyi Biotec); they were selected with the magnetically activated cell sorting (MACS) system (Miltenyi Biotec, Germany) through positive selection using LS columns (Miltenyi Biotec). Single-cell suspensions of CD4⁺ T cells were incubated with fluorescently labeled antibodies specific for CD44, CD62L, CXCR5, and PD1 in staining buffer (PBS with 0.5% BSA) for 20 min at 4°C. Further, the cells were incubated with DAPI to select living cells (DAPI⁻), washed, and T_naïve (CD44⁻CD62L⁺) and T_FH cells (CXCR5⁺PD1⁺) were isolated. Cell sorting was performed using a FACS Influx Sorter (BD Biosciences). The purity of sorted cells ranged from 95% to 98%.