We used the following gating schemes: total leukocytes (CD45.2+), total B cells (CD19+ CD3−), B-2 (CD19+ CD3− B220hi CD5− CD23+), B-1a (CD19+ CD3− IgM+ IgD− B220lo CD5+ CD23−), B-1b (CD19+ CD3− IgM+ IgD− B220lo CD5− CD23−), regulatory B [Breg] cells (CD19+ B220+ CD22+ CD5− IgM+ IgD+), total T cells (CD19− CD3+), Treg cells (CD19− CD3+ CD4+ CD25+), and macrophages (CD19− CD3− CD11b+ F4/80+). Dead cells were distinguished by Live/Dead Fixable Aqua staining (Life Technologies). Baselines for IL-10 EGFP mice were set using age- and diet-matched C57BL/6J mice. For macrophage intracellular cytokine staining, cells were stimulated with LPS (1 μg/mL; Sigma-Aldrich) and brefeldin A (5 μg/mL; BioLegend) overnight and stained using the Cytofix/Cytoperm Kit (BD Biosciences) according to the vendors’ instructions. Data were acquired on an LSR II flow cytometer (BD Biosciences) and analyzed with FlowJo software (Tree Star).
CD16/32, CD3-PacBlue, CD5-PE-Cy5, CD19-PerCP-Cy5.5, CD22-PE, CD23-PE-Cy7, CD25-PE, CD45.2-APC, B220-APC-Cy7, F4/80-PE, F4/80-PErCP-Cy5.5, IgD-PE, and TNF-α–PE antibodies were from BioLegend. IgM-efluor650, CD4-efluor650, and CD11b-efluor605 antibodies were from eBioscience.
CD16/32, CD3-PacBlue, CD5-PE-Cy5, CD19-PerCP-Cy5.5, CD22-PE, CD23-PE-Cy7, CD25-PE, CD45.2-APC, B220-APC-Cy7, F4/80-PE, F4/80-PErCP-Cy5.5, IgD-PE, and TNF-α–PE antibodies were from BioLegend. IgM-efluor650, CD4-efluor650, and CD11b-efluor605 antibodies were from eBioscience.