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. 2019 Aug 21;8:e47015. doi: 10.7554/eLife.47015

Figure 3. Colonization of a microbiota is required for B-1 derived microbiota-reactive IgM.

Figure 3.

(A) Schematic showing the generation of B-1 disparate allotype chimera mice. (B) Serum titers of B-2-derived IgMa and B-1-derived IgMb in 14 wk old B-1 chimeras, as described in (A). (C) Representative flow cytometry plot (left) and quantification (right) of percentage of SYBR+ fecal bacteria bound by B-1-derived serum IgMb and B-2-derived serum IgMa from 14 wk old B-1 chimeras, as described in (A); μMT-/- mouse serum included as a negative control (orange). (D) Representative SYBR staining of germ-free (GF) (left) or SPF μMT-/- (middle) feces, as measured by flow cytometry. Representative flow cytometry histogram plot showing IgM staining of SYBR+μMT-/- fecal bacteria with serum from 7 wk old SPF (black line, no fill) or germ free (GF) mice (gray line, filled) (right). (E) Quantification of percentage of SYBR+μMT-/- fecal bacteria bound by serum IgM from seven wk old SPF (black), GF (gray), or μMT-/- control (orange) mice (right). (F) Serum titers of IgM in 7 wk old SPF (black) or GF (gray) mice, as measured by ELISA. (G-H) Percentage fluorescein-labeled PtC-liposome positive (PtC+) B-1a cells (CD19+CD23–CD43+CD5+) in the (G) peritoneal cavity (PerC) and (H) spleen of 7 wk old SPF (black) or GF (gray) mice, as measured by flow cytometry. Each data point represents an individual mouse (B-C, E-H). Error bars indicate the mean (± SEM). *p<0.05, **p<0.01, and ***p<0.001 (unpaired two-tailed Student's t-test). Data are representative of at least three independent experiments (B-H).