Identification of subsets of splenic natural IgM ASCs differing in their requirements for Blimp-1. (A) Sorting gates for Blimp-1 YFP among B-1a and B-1b splenocytes pregated as live, singlet, dump^neg CD19⁺CD43⁺IgM^hiIgD^lo/neg cells. (B) Mean frequencies ± SD of IgM ASCs among Blimp-1 YFP⁺ (YFP+) and Blimp-1 YFP^neg (YFP-) B-1a and B-1b cells in the spleen (n = 3–8). The dashed line represents the limit of detection. (C, top) Mean maximum IgM spot size ± SD (number of pixels) in each ELISPOT well of sorted Blimp-1 YFP⁺ or YFP^neg B-1a and B-1b cells (n = 3–8) and (C, bottom) representative ELISPOT wells. Numbers indicate total input cells. Results in B and C are representative of two independent experiments. (D) Representative overlay histograms of Blimp-1 YFP among YFP⁺ B-1a and B-1b cells. (E) Mean frequencies ± SD of IgM ASC splenocytes from control and PRDM-1^ΔEx1A mice (n = 11) and (F) control and PRDM-1^ΔEx1A IgM^b peritoneal cavity donor chimeras (n = 4–9) as measured by ELISPOT. Results in E and F are pooled from two independent experiments. (G) B-1–derived IgM ± SD (µg/ml) in supernatants of splenocytes from control and PRDM-1^ΔEx1A B-1 peritoneal cavity donor chimeras as measured by ELISA (n = 3–5). (H, left) Mean maximum IgM spot size ± SD (number of pixels) in each ELISPOT well for control versus PRDM-1^ΔEx1A mice (n = 3) and (H, right) representative ELISPOT wells. Numbers indicate total input cell numbers. Results are representative of two independent experiments. (I) Mean frequencies ± SD of IgM ASCs among splenic CD19^neg IgM⁺ IgD^lo/neg CD43⁺ CD138⁺ B-1PC and CD19⁺ IgM⁺ IgD^lo/neg CD43⁺ B-1 cells (n = 9–10) and of (J) splenic B-1a and B-1b cells (n = 2–4) in control and PRDM-1^ΔEx1A mice. (K, left) Mean maximum IgM spot size ± SD (number of pixels) in each ELISPOT well of sorted Blimp-1 YFP⁺ and YFP^neg B-1a and B-1b cells (n = 2–4) and (K, right) representative ELISPOT wells. Results in I–K are representative of two independent experiments. Statistics in B and C and E–K were done using an unpaired Student’s t test (*, P < 0.05; **, P < 0.005; ***, P < 0.0005).