prv green fluorescent protein gfp foxp3  (Addgene inc)

Journal: Clinical and Experimental Immunology

Article Title: Directed differentiation of regulatory T cells from naive T cells and prevention of their inflammation‐mediated instability using small molecules

doi: 10.1111/cei.13453

Figure Lengend Snippet: Naive T cell activation after ligation of CD3/CD28 in the presence of high levels of interleukin (IL)‐2. (a) After 24 h, naive T cell activation was determined by flow cytometry. (b) The activated naive T cells were compared at 6, 12 and 24 h in the presence of 30, 50, 100 and 200 IU of IL‐2 at a 1 : 1 cell/bead ratio [two‐way analysis of variance (anova)]. (c) Naive T cell activation with different ratios of cells/beads at 200 IU of IL‐2 was determined (one‐way anova). (d) Forkhead box protein P3 (FoxP3) expression level in activated naive T cells at 1 : 3 cells/beads was compared at different concentrations of IL‐2 (two‐tailed unpaired t‐test). All samples were analyzed in duplicate. The results are presented as mean ± standard deviation. *P < 0·05; **P < 0·001; ***P = 0·0002; ****P < 0·0001; five to six donors in the independent experiments.

Article Snippet: RetroNectin (no. T100A/B) and TaqMan were purchased from Takara Biomedical (Shiga, Japan). pRV.green fluorescent protein (GFP) FoxP3 (no. 13250) was purchased from Addgene (Watertown, MA, USA).

Techniques: Activation Assay, Ligation, Flow Cytometry, Expressing, Two Tailed Test, Standard Deviation

Journal: Clinical and Experimental Immunology

Article Title: Directed differentiation of regulatory T cells from naive T cells and prevention of their inflammation‐mediated instability using small molecules

doi: 10.1111/cei.13453

Figure Lengend Snippet: Comparison of ectopic expressions of forkhead box protein P3 (E‐FoxP3), soluble transforming growth factor‐β (S‐TGF) and N‐acetyl puromycin and SR1555 (N‐Ac/SR) protocols for de‐novo generation of regulatory T (Treg) cells. (a) In the three protocols, activated naive T cells responded with reduced levels of CD127 and differentiated towards Treg‐like cells [two‐way analysis of variance (anova)]. (b) Conditioned CD4+ was examined to determine the suppressive activity at different ratios of conditioned CD4+/conventional T (Tconv) cells (two‐way anova). (c) The percentages of FoxP3+ cells and suppressive activity in the selected ratio were compared between the three methods and the control group at day 10 (one‐way anova). (d) Correlations between the percentage of FoxP3+ cells and suppressive activity were determined by linear regression in the three different protocols. All samples were analyzed in duplicate. The results are presented as mean ± standard deviation. (e) Comparison of FoxP3+cytotoxic T lymphocyte antigen 4 (CTLA‐4+)‐generated cells according to the different methods under the inflammatory and non‐inflammatory conditions. (f) Representative flow cytometry plots of FoxP3+CTLA‐4+ cells in the different methods (one‐way anova). *P < 0·05; **P < 0·001; ***P = 0·0002; ****P < 0·0001; three to four donors in the independent experiments.

Article Snippet: RetroNectin (no. T100A/B) and TaqMan were purchased from Takara Biomedical (Shiga, Japan). pRV.green fluorescent protein (GFP) FoxP3 (no. 13250) was purchased from Addgene (Watertown, MA, USA).

Techniques: Comparison, Activity Assay, Control, Standard Deviation, Generated, Flow Cytometry

Journal: Clinical and Experimental Immunology

Article Title: Directed differentiation of regulatory T cells from naive T cells and prevention of their inflammation‐mediated instability using small molecules

doi: 10.1111/cei.13453

Figure Lengend Snippet: The protective and differentiation effects of SR1555 (SR) in the inflammatory condition. Three differentiation protocols were used to evaluate plasticity in de‐novo regulatory T (Treg) cells generated under inflammatory and non‐inflammatory conditions. (a) Differences in Treg generation between the three groups in the inflammatory and non‐inflammatory conditions [two‐way analysis of variance (anova)]. (b) The differences in interleukin (IL)‐17A concentrations between E‐FoxP3 and soluble transforming growth factor‐β (S‐TGF) groups (one‐way anova). (c) The differences in transforming growth factor‐β (TGF‐β) concentration at days 7 and 10 in the ectopic expression of FoxP3 (E‐FoxP3) group in the presence of interleukin (IL)‐6 (unpaired t‐test). (d) The differences in forkhead box protein P3 (FoxP3)+ cells in the inflammatory and non‐inflammatory conditions (one‐way anova). (e) The differences in IL‐10 concentrations between the E‐FoxP3 and soluble TGF‐β (S‐TGF) groups (one‐way anova). (f) Linear regression was used to determine the correlations between FoxP3 and IL‐17A, IL‐10 and interferon (IFN)‐γ. The results are presented as mean ± standard deviation. *P < 0·05; **P < 0·001; ***P = 0·0002; ****P < 0·0001; three to four donors in the independent experiments.

Article Snippet: RetroNectin (no. T100A/B) and TaqMan were purchased from Takara Biomedical (Shiga, Japan). pRV.green fluorescent protein (GFP) FoxP3 (no. 13250) was purchased from Addgene (Watertown, MA, USA).

Techniques: Generated, Concentration Assay, Expressing, Standard Deviation

Journal: Clinical and Experimental Immunology

Article Title: Directed differentiation of regulatory T cells from naive T cells and prevention of their inflammation‐mediated instability using small molecules

doi: 10.1111/cei.13453

Figure Lengend Snippet: Flow cytometry characterization of the generated regulatory T (Treg)‐like cells based on interleukin (IL)‐17A and forkhead box protein P3 (FoxP3). (a) A comparison of the percentages of CD4+IL‐17A+ cells generated by ectopic expression of FoxP3 (E‐FoxP3), soluble transforming growth factor‐β (S‐TGF) and N‐acetyl puromycin and SR1555 (N‐Ac/SR) methods under the inflammatory condition [one‐way analysis of variance (anova)]; three to four donors in the independent experiments. (b) A comparison of the percentages of CD4+IL‐17A+FoxP3+ cells generated by E‐FoxP3, S‐TGF and N‐Ac/SR in the inflammatory condition (one‐way anova); three to four donors in the independent experiments. (c) Representative flow cytometry plots of CD4+IL‐17+ cells in the different methods. (d) Representative flow cytometry plots of CD4+IL‐17+FoxP3+ cells in the different methods; three to four donors in the independent experiments. *P < 0·05; **P < 0·001; ***P = 0·0002; ****P < 0·0001.

Article Snippet: RetroNectin (no. T100A/B) and TaqMan were purchased from Takara Biomedical (Shiga, Japan). pRV.green fluorescent protein (GFP) FoxP3 (no. 13250) was purchased from Addgene (Watertown, MA, USA).

Techniques: Flow Cytometry, Generated, Comparison, Expressing

Journal: Clinical and Experimental Immunology

Article Title: Directed differentiation of regulatory T cells from naive T cells and prevention of their inflammation‐mediated instability using small molecules

doi: 10.1111/cei.13453

Figure Lengend Snippet: SR1555 (SR)‐enriched regulatory T (Treg) cells and increased the capacity for T cell suppression. (a) A comparison of Treg‐like cells generated by the ectopic expression of forkhead box protein P3 (E‐FoxP3) and soluble transforming growth factor‐β (S‐TGF) protocols under the non‐inflammatory and inflammatory conditions, with and without SR. (b) Flow cytometry analysis of the conditioned T cells to determine the percentages of the Treg‐like cell population according to CD4+CD25+CD127dim/‐ (c) Relative expression of exogenous and endogenous FoxP3 in the non‐inflammatory and inflammatory conditions, with and without SR [one‐way analysis of variance (anova)]. (d) Flow cytometry analysis of the conditioned T cells to determine the percentages of the Treg‐like cell population according to CD4+CD25+FoxP3+. (e) Linear regression to determine the correlation between FoxP3 and suppressive activity in the E‐FoxP3 and S‐TGF subgroups. The results are presented as mean ± standard deviation. *P < 0·05; **P < 0·001; ***P = 0·0002; ****P < 0·0001; three to four donors in the independent experiments.

Article Snippet: RetroNectin (no. T100A/B) and TaqMan were purchased from Takara Biomedical (Shiga, Japan). pRV.green fluorescent protein (GFP) FoxP3 (no. 13250) was purchased from Addgene (Watertown, MA, USA).

Techniques: Comparison, Generated, Expressing, Flow Cytometry, Activity Assay, Standard Deviation

Journal: Clinical and Experimental Immunology

Article Title: Directed differentiation of regulatory T cells from naive T cells and prevention of their inflammation‐mediated instability using small molecules

doi: 10.1111/cei.13453

Figure Lengend Snippet: The combination of small molecules converts naive T cells into regulatory T (Treg) cells that show high suppressive activity in the inflammatory condition. (a) Analysis of Treg‐specific demethylation region (TSDR) demethylation in the generated Treg‐like cells by different methods under inflammatory and non‐inflammatory conditions. nTreg was used as the control [one‐way analysis of variance (anova)]. (b) The relative expressions of receptor‐related orphan receptor gamma (RORγ)‐t and forkhead box protein P3 (FoxP3) in Treg‐like cells generated by the different methods in the inflammatory condition; two to three donors in the independent experiments. nTreg was used as the control (one‐way anova). (c) De‐novo generation of Treg cells using small molecules in the presence of interleukin (IL)‐6 was compared in the three N‐acetyl puromycin and SR1555 (N‐Ac/SR) subgroups (one‐way anova). (d) IL‐17A levels were measured in the different N‐Ac/SR subgroups after 10 days in the inflammatory condition (one‐way anova). (e) CD25+FoxP3+ cells were identified by flow cytometry gating on CD4+ cells after 10 days in the inflammatory condition. (f) Suppressive activity was analyzed by carboxyfluorescein succinimidyl ester (CFSE) after 10 days. A comparison was made between the different N‐Ac/SR subgroups in the inflammatory condition. The results are presented as mean ± standard deviation. *P < 0·05; **P < 0·001; ***P = 0·0002; ****P < 0·0001; three to four donors in the independent experiments.

Article Snippet: RetroNectin (no. T100A/B) and TaqMan were purchased from Takara Biomedical (Shiga, Japan). pRV.green fluorescent protein (GFP) FoxP3 (no. 13250) was purchased from Addgene (Watertown, MA, USA).

Techniques: Activity Assay, Generated, Control, Flow Cytometry, Comparison, Standard Deviation

Journal: Clinical and Experimental Immunology

Article Title: Directed differentiation of regulatory T cells from naive T cells and prevention of their inflammation‐mediated instability using small molecules

doi: 10.1111/cei.13453

Figure Lengend Snippet: Different methods for regulatory T cell (Treg) differentiation in the co‐culture of naive T and nTreg cells. (a) In the co‐culture groups, CD25+forkhead box protein 3 (FoxP3+) cells were compared in two differentiation groups, soluble transforming growth factor‐β (S‐TGF) group and the N‐acetyl puromycin and SR1555 (N‐Ac/SR) group, under inflammatory and non‐inflammatory conditions [one‐way analysis of variance (anova)]. (b) In the co‐culture groups, the levels of interleukin (IL)‐17A, IL‐10 and interferon (IFN)‐γ were measured in the two groups after 10 days in the non‐inflammatory condition (one‐way anova). (c) Suppressive activity was analyzed by carboxyfluorescein succinimidyl ester (CFSE) after 10 days. A comparison was made between the N‐Ac/SR and S‐TGF protocols (one‐way anova). The results are presented as mean ± standard deviation. *P < 0·05; **P < 0·001; ***P = 0·0002; ****P < 0·0001; three to four donors in the independent experiments.

Article Snippet: RetroNectin (no. T100A/B) and TaqMan were purchased from Takara Biomedical (Shiga, Japan). pRV.green fluorescent protein (GFP) FoxP3 (no. 13250) was purchased from Addgene (Watertown, MA, USA).

Techniques: Co-Culture Assay, Activity Assay, Comparison, Standard Deviation