Fig. 2: Linking IgG secretion to cell surface markers and intracellular machinery using flow cytometry.

a Schematic of the staining format used to analyze single B cell IgG secretion and cell surface markers by flow cytometry. Created with BioRender.com. b Representative flow cytometry density scatter plots for surface markers to identify populations of ASCs from active B cells using CD19 and CD38 staining. IgA cells, IgM cells, and ASCs not producing either IgA or IgM (double negative, DN) were gated based on IgA and IgM staining. Fluorescence histograms of IgG secretion signal for the various identified gates and empty nanovials containing no cells. c Contour plot for CD38 and CD138 staining in the DN population and fluorescence histograms of IgG signal from populations within these gates. The dot graph presents the % of IgG secretors from each subset (n = 8 independent analyses, from four donors). Data were presented as mean ± SD. P values were calculated using a paired one-way analysis of variance with a Tukey’s test for multiple comparisons. Source data are provided as a Source Data file. d Histogram of IgG levels for IgG non-secretors and IgG secretors from different ASC subsets. The linked dot graph presents the mean fluorescence intensity of IgG secretors from each ASC subset. The p value was calculated using a two-sided paired t-test (n = 7 independent analyses, from three donors). Source data are provided as a Source Data file. e Imaging cytometry results confirm that IgM+ cells have low levels of secreted IgG, CD138loCD38++ populations have intermediate levels of secreted IgG, and CD138hiCD38++ have the highest levels of secreted IgG. f Imaging flow cytometry gating strategy for IgG quantification by cell subtype (upper). The dot graph presents the MFI of IgG from each ASC subset (n = 3 independent analyses from three donors). P values were calculated using paired one-way analysis of variance with Tukey’s test for multiple comparisons. Source data are provided as a Source Data file.