Figure 4.

Reagent panel selection. Shown are contour plots with outliers for staining of SEB-stimulated PBMC with 10 different reagent panels that afforded robust T lymphocyte subset separation. Cells were gated on lymphocyte FSC vs. SSC, exclusion of dead cells, and staining for CD3. While similar frequencies of CD4 and CD8 T cells were measured with panels 11, 12, 14, 24, 28, and 29, a higher fraction of CD4⁺CD8⁺ double positive (DP) T cells was identified in panels 23, 26, 27, and 30 (shown in red). The increased frequency of DP cells are likely due to unique and undesirable spectral interactions, or to the presence of the OKT8 CD8 monoclonal antibody, and preclude reliable measurement of CD4⁺ T cells in these four panels.