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. Author manuscript; available in PMC: 2014 Aug 1.
Published in final edited form as: J Immunol. 2013 Jul 3;191(3):1260–1275. doi: 10.4049/jimmunol.1300770

Figure 4. Distribution of splenic and peritoneal B-cell subsets in C57BL/6 (WT) and 4E10/2F5 complete (VH+/+ × VL+/+) KI strains.

Figure 4

A. FACS contour plot histograms (representative of three experiments) showing frequencies of total (singlet/live/lymphocyte, B220+CD19+) B-cells in spleens of 6-12 wk naïve mice. B. Statistical analysis of total splenic B-cell percentages (based on flow cytometric analysis shown in Fig. 4A, with each circle, square and triangle representing an individual 6-12 wk C57BL/6 (WT), 2F5 complete KI, and 4E10 complete KI mouse, respectively. Horizontal lines represent averages for each group. Significance values were determined using a two-tailed student test. NS: P>0.05, *P≤0.05, **P ≤0.01, ***p ≤0.001. C. Representative FACS contour plot histograms showing analysis of splenic B-cell subsets derived from 8-12 wk female naïve mice, fractionated and annotated as indicated in Fig. 2B. D. Statistical analysis of splenic B-cell subsets from individual WT or 2F5/4E10 complete KI mice, represented either as frequencies of newly formed/transitional (CD21−CD23−), MZ (CD23intCD21hi), and Mat (CD23hiCD21int) subsets (upper panel) or transitional (B220+CD93+)/Mat+MZ (B220+CD93−) ratios (lower panel). E. In vivo turnover of the predominant, transitional splenic B-cell subset in 4E10 complete KI mice. 8 wk naïve female naïve 4E10 complete KI or control C57BL/6 (WT) mice (lower and middle panels, respectively) were continuously labeled with BrdU for 4 days, prior to flow cytometric enumeration of the percent of BrdU+ B-cells within singlet/live, lymphocyte-gated transitional (B220+CD93+) and mature/MZ(B220+CD93−) splenic B-cell subsets, as indicated in the shaded histograms. Also shown as negative controls are C57BL/6 (WT) mice without BrdU treatment (top panel). Data is representative of two mice/group. F. Representative FACS contour plot histograms showing analysis of peritoneal B-cell subsets as revealed by surface staining of live lymphocytes with B220 and either CD5 (upper row) or IgM (bottom row), with numbers in each gate indicated, and B-cell subsets indicated in the upper row are defined as for Fig. 2C. G. Analysis of total serum Ig subclass levels in C57BL/6 (WT) mice and 2F5/4E10 complete KI strains. Serum samples were collected from 6-12-wk naive mice, and Ig subclass concentrations were determined by Luminex analysis. Each symbol represents an individual mouse and horizontal lines represent averages for each group. H. Flow cytometric analysis of sIg densities in total (singlet/live/lymphocyte, B220+CD19+) splenic B-cells from 6-12 wk naïve mice, as measured by Median Fluorescence Intensity (MFI) quantifications of surface κL chain (left panel) or surface IgM (right panel) expression.