1. 1 2 3 4 5 Suppl. Fig. 3 0.06x106 Culture of Irradiated A-CFSE
PKH26
CFSE
70.8%
0.06x106
CD4-ECD
CFSE
CD4 Responders
11.3%
FOXP3-PC5
CFSE
2.6%
CD4+CD25High FOXP3+
FOXP3-PC5
CD25-PC7
Culture of Irradiated A-CFSE
Irradiated B-PKH26 +
Remove PKH26+ Stimulators
Gate on CD4+ Responders (Irradiated)
FOXP3+ in CD4+ Responders (Irradiated)
CD25HighFOXP3+ Cells in CD4+ Responders (Irradiated)
Size & Granularity
&
Suppl. Fig. 3
Supplemental Digital Content 5 - Figure 3: Control Culture: 5x105 CFSE-labeled and x-irradiated PBMC from the MLR responder were cultured with 5x105 PKH26 labeled and x-irradiated stimulator PBMC. After 7 days, 5-color flow cytometric assays were performed. The PKH26 positive cells (any remaining) were gated out and then the CD4+ cells were analyzed for CD25 and  FOXP3 expression. The source of the CFSE and PKH26 double positive cells (2nd from left) are unknown; possibly they are crenated aggregates, and are distributed throughout the FS vs. SS scatter-plot. Note that the number of CD4+ cells remaining after 7 days in culture  (shown in red on top of the 2nd column density-plot) are far fewer (0.06x106) than in the MLR cultures shown in Fig. 3A & B (1.1x106 & 1.8x106 respectively). Additionally, even in the few remaining cells, the CD4+ cells and FOXP3+ or CD25+ subsets are significantly diminished. Taken together, these data suggest that the irradiated stimulator cells in the MLR cultures do not contribute to the subsets analyzed in flow cytometry.