We are conducting flow cytometry to detect IL-10 secretion in B cells, specifically regulatory B cells (Bregs). For this purpose, we utilize either whole blood (100 μl) or isolated PBMCs (1 million cells/ml of RPMI-1640+10% FCS). These samples are stimulated with or without CpG (ODN 2006; TLR9 agonist [2-6 μg/ml]) for various time points (16h, 24h, 42h, and 72h). We add DURActive1 (Beckman Coulter), a dry stimulation mix containing PMA, ionomycin, and Brefeldin A, to the reaction during the last five hours of incubation.We also include non-treated and No CpG (samples solely incubated with DURActive1 for 5h) control samples. We utilize a mixture of different fluorescently labelled antibodies to detect IL-10 , Lymphocytes (CD45+ cells), T cells (CD5+ cells), and Bregs (CD19, CD38, CD27, CD11b, CD73, CD39, CD1d, CD24, CD71).

The maximum stimulation effect we have achieved with this setup is approximately 2-4 fold, considering the basal level of IL10+ B cells is below 1%. Surprisingly, CpG-treated samples exhibit lower IL-10 expression compared to samples treated solely with DURActive1, with only a slight increase compared to the non-treated control.

Non-B cells (CD45+CD19-) display a robust response to DURActive1 treatment, exhibiting a 20-30 fold increase in IL-10 expression compared to the non-treated control. However, the addition of CpG does not alter this effect. As the stimulation appears to be effective for non-B cells, we believe the sample preparation and staining protocol for flow cytometry are not of concern.

we actively explore methods to efficiently induce IL-10 expression in B cells. Any feedback or suggestions you may have to improve the current method outlined above or alternative approaches that demonstrated effectiveness, would be greatly appreciated.