Using a 10–color human B cell panel, it is possible to zoom in on B cells and more deeply phenotype this population of interest. We have made use of our pre–optimized, experimentally verified 35–color immunophenotyping panel and generated this 10–color backbone panel. With our B cell backbone, you can use the most common markers to identify B cells and then add the deeper markers based on the hypothesis you are testing. In addition to the 8 markers from the original 35–color panel, we have included 2 additional markers CD10 and CD21 to further recognize transitional B cells and memory B cell subpopulations respectively.
With these 10 markers, subpopulations of peripheral blood human B cells can be recognized including transitional B cells, plasmablasts, memory populations of class–switched B cells as well as marginal zone and naïve populations of unswitched B cells. Since the reduced B cell panel is predominantly focused on identifying subpopulations of B cells, there are plenty of spaces in the complete panel to include additional phenotyping markers. In Figure 1, B cells are recognized based on CD19 and CD20 expression. CD10 is used to identify transitional B cells that predominantly express IgD and CD38.
After excluding CD10+ transitional B cells, C38 and CD24 can be used to separate plasmablasts from non–plasmablasts. Non–plasmablasts are divided into unswitched and class–switched B cells based on IgM and IgD expression. Non–class–switched more naïve B cells were split into activated naïve and resting naïve populations based on patterns of CD38, CD21 and CD24 expression. Class–switched B cells were allocated into activated memory (AM), resting memory (RM), tissue–like memory (TLM) and intermediate memory (IM) populations using CD21 and CD27 expression.
Figure 1. Gating of human peripheral blood B cell panel with singlets (top left), live cells (top second from left) lymphocytes (top third from left), and then CD19+ CD20+ B cells (top second from right). Transitional B cells were identified based on CD10 expression (top right) and were predominantly CD38+ IgD+ (middle right). Next, plasmablasts were separated from non–plasmablasts based on CD38 hi CD24– gate (middle left). Class–switched B cells were identified as IgD– IgM– (middle first from left) and activated memory (AM), resting memory (RM), tissue–like memory (TLM) and intermediate memory (IM) were gated based on CD21 and CD27 expression (bottom left). The unswitched B cells were also gated into naïve and marginal zone (MZ) based on IgD vs CD27 expression (middle second from left). The unswitched naïve B cells were then further subdivided into activated naïve and resting naïve populations based on expression of CD38, CD21 and CD24 as shown (lower right 3 plots) (Click image to enlarge).
Human PBMCs were stained and acquired on a 5–Laser Cytek Aurora with standard configuration, using the Cytek assay settings. CellBlox Blocking Buffer and Brilliant Stain Buffer were added to the antibody cocktail to block non–specific cell binding to NovaFluor dyes and polymer dye–dye interactions, respectively. Cells were stained with antibodies (table 1) for at least 30 minutes at 4°C in the dark and fixed with 2% paraformaldehyde. Antibodies were titrated to determine optimal staining concentrations for cells. Single colors were generated on both cells and beads with the most optimal control (cells by default, beads if needed) used for unmixing raw data files. Spectral unmixing was performed using Cytek SpectroFlo software version 3.1.0. Analysis was performed using BD FlowJo version 10.8.1.
Table 1. Markers from 10–color 5–laser Cytek Aurora panel grouped by cell type expression for B cells. 8 B cell specific markers from an experimentally verified 35–color broad human immune panel were selected and 2 additional markers (see * in the table below) were included to allow for identification of transitional B and B cell memory subpopulations. This panel serves as a B cell backbone that can be used as is or allows for the inclusion of additional antibodies further phenotype B cell subpopulations. Primary markers refer to lineage markers that define populations and are typically highly and clearly expressed. Secondary markers refer to higher density markers with more continuous expression that typically subphenotype populations. Tertiary markers are typically expressed at lower levels.
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