Flow Cytometric Analysis of B Cell Populations

Flow cytometric analysis of B cell populations was performed using our previously described gating strategies8 (link)15 (link)16 (link)17 (link). Following red cell lysis, cells were counted and stained with a cocktail of antibodies for the detection of the indicated populations using anti-CD19-AF700, anti-CD23-PE/Cy7, anti-CD21-E450, anti-CD43-PE/Cy7, anti-GL7-FITC, anti-CD138-PE, anti-B220-E450, anti-B220-APC, anti-IL-10-APC, anti-CD3-FITC, anti-CD4-PerCP and anti-CD8-PE/Cy7 antibodies from eBioscience and Biolegend. Plasma cells were phenotyped as (Dump (CD3, CD4, CD8, CD11b, GR1 & CD11c)⁻ CD138⁺CD19⁻B220⁻) with the Dump⁺ markers identified using biotinylated antibodies and svPE. Alternatively, splenocytes were stimulated with 50 ng/ml PMA (Sigma-Aldrich, UK) plus 500 ng/ml ionomycin (Sigma-Aldrich, UK) and 10 μg/ml LPS (E. coli O111:B4, Sigma-Aldrich, UK) for 1 h before addition of 10 μg/ml Brefeldin A (Sigma-Aldrich, UK) and cells were then incubated for a further 5 h at 37 °C with 5% CO₂. Cells were fixed and washed several times in permeabilization buffer before anti-IL-10 was added in permeabilization buffer and incubated at 4 °C for 30 minutes. Cells were then washed three times with permeabilization buffer and finally with FACs staining buffer before data were acquired using a BD LSR II flow cytometer and analysis undertaken by FlowJo software (TreeStar).