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. 2021 Feb 22;10:e57417. doi: 10.7554/eLife.57417

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© 2021, Zhou et al

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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Figure 2. — (A and B) Treg proliferation as measured by CellTrace signal following 3 days of culture with αCD3ε and all combinations of IL-2 and IL-4, with division and proliferation indices indicated. N = 3 for all bar graphs and histograms are representative. (C) IL-10 expression of purified Tregs cultured for 3 days with αCD3ε and the designated cytokines, and gated by CellTrace generation, as measured by flow cytometry (panel A). N = 3. The IL-10 expression of all IL-2/IL-4-induced Treg generations are statistically significant (p<0.05) compared to the other cytokine-stimulated conditions. (D) Proliferation and viability of purified IL-10⁺ and IL-10^- Tregs cultured for 3 days with αCD3ε and both IL-2 and IL-4, as flow cytometry analysis of CellTrace and Sytox Red signal. The histograms are representative. N = 3. (E and F) Proliferation of purified Tregs cultured for 3 days with αCD3ε and all cytokine combinations, gated on IL-10^+/- expression, as analyzed by flow cytometry. N = 3 for all bar graphs and histograms are representative. (G) Division index of wild type (FoxP3^creYFP) and IL-10 cKO (IL-10^fl/fl× FoxP3^creYFP) purified Tregs that were stimulated with the indicated conditions for 3 days, as analyzed by flow cytometry. N = 3 for all bars. (H) Cell count of IL-10⁺ Tregs following 3–7 days of culture with αCD3ε and all combinations of IL-2 and IL-4, as analyzed by flow cytometry. N = 3. The cell counts of all IL-2/IL-4-induced conditions are statistically significant (p<0.05) compared to the other cytokine-stimulated conditions. For all panels, mean ± SEM are indicated. *p<0.05, **p<0.01, ***p<0.001.

Figure 2—figure supplement 1. — (A and B) Treg proliferation as measured by CellTrace signal following 3 days of culture with αCD3ε and all combinations of IL-2 and IL-4, with division and proliferation indices indicated. N = 3 for all bar graphs and histograms are representative. (C) IL-10 expression of purified Tregs cultured for 3 days with αCD3ε and the designated cytokines, and gated by CellTrace generation, as measured by flow cytometry (panel A). N = 3. The IL-10 expression of all IL-2/IL-4-induced Treg generations are statistically significant (p<0.05) compared to the other cytokine-stimulated conditions. (D) Proliferation and viability of purified IL-10⁺ and IL-10^- Tregs cultured for 3 days with αCD3ε and both IL-2 and IL-4, as flow cytometry analysis of CellTrace and Sytox Red signal. The histograms are representative. N = 3. (E and F) Proliferation of purified Tregs cultured for 3 days with αCD3ε and all cytokine combinations, gated on IL-10^+/- expression, as analyzed by flow cytometry. N = 3 for all bar graphs and histograms are representative. (G) Division index of wild type (FoxP3^creYFP) and IL-10 cKO (IL-10^fl/fl× FoxP3^creYFP) purified Tregs that were stimulated with the indicated conditions for 3 days, as analyzed by flow cytometry. N = 3 for all bars. (H) Cell count of IL-10⁺ Tregs following 3–7 days of culture with αCD3ε and all combinations of IL-2 and IL-4, as analyzed by flow cytometry. N = 3. The cell counts of all IL-2/IL-4-induced conditions are statistically significant (p<0.05) compared to the other cytokine-stimulated conditions. For all panels, mean ± SEM are indicated. *p<0.05, **p<0.01, ***p<0.001.