BM B-1PC, but not B-1 cells, require Blimp-1 for maximal IgM secretion. (A) Gating for total and Blimp-1 YFP^neg (YFP-) B-1a and B-1b BM cells, pregated on live, singlet, dump^neg, CD19⁺CD43⁺IgM^hi IgD^lo/neg cells. (B) Mean frequencies ± SD. IgM ASCs among total and Blimp-1 YFP^neg B-1a and B-1b cells in the BM (n = 4). The dashed line represents the limit of detection. Results are representative of two independent experiments. (C) Prdm1, irf4, zbtb20, and igj (J chain) mRNA expression (Log) ± SD by FACS-sorted follicular B cells (FO B), BM B-1 cells, and B-1PCs, normalized to ubc (n = 3). Results are combined from two independent experiments. (D) Fluorescent imaging of (left to right) FACS-sorted B-1PCs (first column) and BM B-1 cells either showing (second column) or not showing (third column) intracellular IgM staining, all taken from Blimp-1 YFP reporter mice and B-1PCs (fourth column) taken from C57BL/6 mice. Top to bottom: differential interference contrast images, Blimp-1 YFP (green), intracellular IgM (red), and merged images. Arrows indicate intracellular IgM vesicles. Smaller blue boxes indicate the magnified area found in the larger blue box in the same image. The scale bars (25 µm) in the top left image apply to all images. FMO, fluorescence minus one control stains for intracellular IgM (fifth column). C57BL/6 B-1PCs serve as FMO for YFP expression and are stained with anti–GFP/YFP-FITC. Images are representative of multiple cells from two independent experiments. Statistics in B and C were done using an unpaired Student’s t test (*, P < 0.05; **, P < 0.005; ***, P < 0.0005).