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For cell proliferation, purified T cell was stained with CFSE and detected cell division by flow cytometry after 5 days of incubation. 2.6. Western Blot and ELISA Cells were collected after 5 days induced by 1,25(OH)2D or controls for VDR analysis by WB. For cytokine measurements, cell culture supernatants were collected from 24?h to [http://www.selleck.co.jp/products/atezolizumab.html http://www.selleck.co.jp/products/atezolizumab.html Atezolizumab mouse selleck chemical Atezolizumab] 7?d and diluted for the measurement of cytokines IL-10, IL-17, and IFN-�� quantitatively using enzyme-linked immunosorbent assay (ELISA) kits (Multisciences Biotech), according to the manufacturer's instructions. The detection limit of IL-10, IL-17, and IFN-�� was 0.7?pg/mL, 15?pg/mL, and 15?pg/mL, respectively. 2.7. Statistics The statistical evaluation was performed with GraphPad Prism (version 5.0, GraphPad Software, CA). Values are shown throughout the paper as mean �� SEM except for the patients and HD age, which is shown as mean �� SD. A Student t-test was used to analyze the differences between the groups and one-way ANOVA was initially performed to determine whether an overall statistically significant change existed before using the two-tailed paired or unpaired Student t-test for normal distributed data. In the case of significant differences between subgroups, post hoc analyses were based on the Tukey test (normal distributed data) or on the Mann/Whitney U test. Pearson's correlation coefficient (normal distributed data) and Spearman's rank correlation coefficient (nonnormal data) were used to assess interrelationships. TGF-beta inhibitor A P value of C646 1.8%, while the percentages in patients with CHF were from 0.7% to 7.2%. The frequencies of Th17 cells were markedly higher in patients associated with clinical stage (NYHA II 1.98 �� 0.24%, NYHA III 2.29 �� 0.28%, and NHYA IV 1.68 �� 0.29%) compared with the healthy donors (0.99 �� 0.07%) and elder donors (0.95 �� 0.09%) (P