Purification of Murine IL-10 ⁺ B Cells for Analyses of Biological Functions and Transcriptomics

B10 cells are the most frequently investigated subset of Breg cells, capable of suppressing immunity through the expression of the immunosuppressive cytokine IL-10. B10 cells are enriched in phenotypically diverse B-cell subsets. Recently, CD9 was identified as a marker of B10 cells in mice (human B10 cells have a separate set of markers that do not overlap with murine B10 cells). Together with a combination of other B10 markers, CD9 can be used to distinguish both mature and immature B10 cells from nonregulatory B cells and support selective purification of B10 cells. Here we provide five methods for the characterization and activity evaluation of CD9⁺ B cells. The first method is used for the preparation of leukocytes, the second and third are used for the characterization of CD9⁺ B cells, while the last two methods serve to evaluate CD9⁺ B-cell activities. Finally, we detail the purification of RNA from B10 cells and the performance of transcriptomic assays.