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. 2019 Aug 21;8:e46997. doi: 10.7554/eLife.46997

Figure 2. CD5+ B-1 cells decrease CD5 expression after LPS stimulation in vitro.

(A) Representative FACS plots (left) and mean percentage ± SD (right) of CD5+ and CD5- B-1 cells after FACS-purified peritoneal cavity CD19+ CD23- CD5+ B-1 cells were cultured with or without 10 µg/ml LPS for 3 days (n = 18). (B) CD5 expression on FACS-purified Efluor 670-stained proliferating peritoneal cavity CD5+ B-1 cells stimulated with LPS compared to CD5 FMO (fluorescence minus one) control. (C) Mean CD5 MFI ± SD, determined by flow cytometry, (D) mean Log(cd5 mRNA expression) ± SD, determined by qRT-PCR, and (E) mean IgM secretion ± SD (µg/ml), determined by ELISA, after purified peritoneal cavity CD5+ B-1 cells were cultured for indicated times with LPS (n = 3–4 per time and data point). Results are combined from 4 (A), or are representative of >5 (B), and 2 (C-E) independent experiments, respectively. Values in (C–E) were compared using an unpaired Student’s t test (*=p < 0.05, **=p < 0.005, ***=p < 0.0005, ****=p < 0.00005).

Figure 2.

Figure 2—figure supplement 1. CD5- B-1 cells do not survive better or proliferate more compared to CD5+ B-1 cells.

Figure 2—figure supplement 1.

CD5+ and CD5- B-1 cells isolated by FACS from the body cavities of wild type C57BL/6 mice were cultured separately or mixed as indicated for 72 hr in the presence or absence of LPS. (A) Sample FACS plots from cultures with or without LPS of purified CD5+ B-1 cells mixed or not with indicated percentages of CD5- B-1 cells. (B) Mean CD5 MFI ± SD (left) and mean percentage ± SD of CD5+ cells (right) of cultures in A (n = 3). (C) CD5+ (green) and CD5- (purple) B-1 cells were each labeled with either CFSE or Efluor670. Dyes used to label each population were switched for repeated experiments. (D) Mean percentage ± SD of live cells, or (E) of divided cells, (F) mean number of divisions ± SD among cells that had divided, and (G) mean percentage ± SD of cell numbers on day three compared to input numbers for CD5+ and CD5- cells cultured with LPS (n = 8). Results are representative of 2 (A–B) or 4 (D–G) independent experiments. Values in (B) and (D–G) were compared using an unpaired Student’s t test (*=p < 0.05, ****=p < 0.00005).