Suppl. Fig x106 Culture of Irradiated A-CFSE

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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.