Figure 33.

Quantification of human CD25^hhCD127^lo Tregs using whole blood. (A) Count beads were gated based on SSC properties and CD3 expression. (B-E) After the exclusion of the beads, CD45⁺ whole blood cells were selected, doublet cells were excluded, and total lymphocytes were gated based on SSC and FSC properties. (F-H) From CD3⁺ T cells, CD4⁺CD8⁻ T cells were selected. Within the latter gate, CD25⁺CD127^lo Tregs and T conventional cells were identified. The Trucount tubes contain a number of beads that is used to calculate the absolute counts of the Tregs per μL based on the equation: (Number of positive Treg events/Number of bead events) *(Number of beads per tube/Test blood volume). Data were collected on a BD Fortessa X20 cytometer (see Table 34).