Fig. 5.

Fluorescence-minus-one control (FMO). PBMCs from the same donor were stained. Mononuclear cells were selected using forward and side light scatter parameters together with CD45 and viability staining. CD3 expression was used to identify T cells, and subsets were identified by CD4 and CD8 expression. The boxes in red indicate the position of the gating based on FMO. a Gated on T cells, left panel shows a bivariate dot plot of the complete staining for CD4 versus CD8; middle panel shows FMO control of CD8 Qd605; and right panel shows FMO control of CD4 V450. b Gated on CD4⁺ T cells, left panel shows bivariate dot plot of the complete staining for regulatory T cell identification; middle panel shows FMO control of CD25 PE-Cy7; and right panel shows FMO control of Foxp3 PE. c Gated on T cells, left panel shows bivariate dot plot of the complete staining for CD27 versus CD39; middle panel shows FMO control of CD27 Qd655; and right panel shows FMO control of CD39 AF488. Note, that with CD25, the FMO identifies all positive cells, not only the bright cells characteristic of T regulatory cells, thus illustrating the subjectivity of place gates even with the use of FMO controls