Splenic and VAT B cell subsets were identified by the following membrane markers. Follicular (FO): CD19+AA4.1-CD43-CD23+CD21int; Marginal Zone (MZ): CD19+AA4.1-CD43-CD23low/negCD21hi; Age-associated B cells (ABC): CD19+CD43-AA4.1-CD23-CD21-. AA4.1 (CD93) is the marker of transitional B cells. CD43 is the marker of B1 B cells. Both transitional and B1 B cells are excluded by cell sorting to obtain only B2 B cells. Plasma cells were evaluated by membrane expression of CD138 in cultured cells.
B cells were membrane stained for 20 min at rt with Live/Dead detection kit (ThermoFisher) and with the following antibodies: PacBlue-conjugated anti-CD45 (Biolegend 103125), APC-Cy7-conjugated anti-CD19 (BD 557655), PE-Cy7-conjugated anti-AA4.1 (eBioscience 25-5892-81), APC-conjugated anti-CD43 (BD 560663), FITC-conjugated anti-CD21/CD35 (BD 553818), and PE-conjugated anti-CD23 (BD 553139). To evaluate plasma cell frequencies, splenic B cells were membrane stained with PE-conjugated anti-CD138 antibody (BD 553714). Cells were then fixed with BD Cytofix (BD 554655). Up to 105 events in the lymphocyte gate were acquired on an LSR-Fortessa (BD) and analyzed using FlowJo 10.0.6 software.
B cells were membrane stained for 20 min at rt with Live/Dead detection kit (ThermoFisher) and with the following antibodies: PacBlue-conjugated anti-CD45 (Biolegend 103125), APC-Cy7-conjugated anti-CD19 (BD 557655), PE-Cy7-conjugated anti-AA4.1 (eBioscience 25-5892-81), APC-conjugated anti-CD43 (BD 560663), FITC-conjugated anti-CD21/CD35 (BD 553818), and PE-conjugated anti-CD23 (BD 553139). To evaluate plasma cell frequencies, splenic B cells were membrane stained with PE-conjugated anti-CD138 antibody (BD 553714). Cells were then fixed with BD Cytofix (BD 554655). Up to 105 events in the lymphocyte gate were acquired on an LSR-Fortessa (BD) and analyzed using FlowJo 10.0.6 software.