Protocol for improved resolution of plasma cell subpopulations by flow cytometry

Abstract

Plasma cells are rare cells that have been notoriously difficult to detect by flow cytometry. New advances have described B220+ CD138+ plasma cells in the bone marrow that are particularly difficult to distinguish between CD138 intermediate B220+ developing B cells. Herein we describe a novel method for detecting plasma cells in the bone marrow using a combination of CD138 and Sca-1 staining.

Keywords: B cells; B220; Blimp-GFP; CD138; Flow cytometry; Plasma cells.

Figure 1

Flow cytometric analysis of mouse BM, splenic, and Peyer's patch plasma cells. (A) BM cells from an adult B6.Blimp^GFP/+ were isolated and stained for analysis by flow cytometry. Cells were pre-gated based on size using FSC-A by SSC-A then doublets were excluded by gating on FSC-W by SSC-W. Live cells were analyzed by gating on Zombie Aqua negative cells. Gating on IgD^- and Dump^- cells enriched for cells that include plasma cells. Plasma cells make up the CD138^high B220^+/- population and developing B cells are found in the CD138^low B220⁺ population. Both cell populations can be further analyzed for Blimp-GFP expression to confirm plasma cell identity. (B) Cells are pre-gated as in (A) to reveal a CD138^high Sca-1⁺ population. Further analysis of these cells reveals Blimp-GFP expression and both immature B220⁺ and mature B220^- plasma cells. (C) Splenic plasma cells were analyzed by pre-gating as in (A) then examining the CD138^high Sca-1⁺ cells for Blimp-GFP expression. (D) Representative Peyer's patch cells gated as in (C). Flow cytometric graphs shown are representative of >5 independent experiments of 3 or more mice per group.

Figure 2

Sodium azide decreases the intensity of CD138 staining of Blimp-GFP⁺ plasma cells. (A) BM cells were prepared in FACS buffer with 0, 0.005, 0.01, or 0.05% sodium azide. Cells were then stained for flow cytometry as in Figure 1 and gated on viable IgD^- Dump^- cells. Plasma cells were first gated on the Blimp-GFP⁺ Sca-1⁺ before examining CD138 staining intensity. Shown are representative plots from cells prepared with 0 versus 0.05% sodium azide. (B) Mean fluorescence intensity of CD138 derived from the Blimp-GFP⁺ Sca-1⁺ plasma cell population prepared with the indicated concentration of sodium azide. Results are representative of 3 independent experiments with 3 or more samples per group. Error bars represent the standard error of the mean. *, p<0.001 from control as determined by one way ANOVA with a Bonferroni post-test.