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p.) correolide C (n?=?5)] and day 35 [correolide C?+?30?days tacrolimus?+?ALS (n?=?4)], respectively. Skin samples were stored in dispase (1?mg/ml, Roche Diagnostics GmbH, Vienna, Austria) overnight. After washing with complete medium, (10% FCS and 1% pen/strep in RPMI 1640; LONZA, Basel, Switzerland) skin samples were digested in specific collagenase D (1?mg/ml; Roche) for 1?h at 37?��C. Next, the cell suspension was strained through a Nylon cell strainer and washed twice. For lymphocyte isolation, the cell suspension was slowly placed on histopaque (histopaque 1083; Sigma-Aldrich Company Ltd, Irvine, Ayrshire, UK) and centrifuged http://www.selleckchem.com/products/z-vad-fmk.html at 800?rcf for 30?min at 20?��C without breaks. The white blood cell ring fraction was transferred into PBS, washed three times and used for flow cytometry analysis. The spleen was stored in complete medium overnight. The tissue was homogenized by straining through a Nylon cell strainer and then continued with the protocol described above. Heparinized peripheral blood was slowly placed on histopaque and continued http://www.selleck.cn/products/BIBW2992.html with the protocol described above. For three/four-color flow cytometry cell suspensions were incubated at 4?��C for 30?min with appropriate dilutions of directly labeled monoclonal rat/mouse antibodies against CD3 (PE, G4.18), CD4 (FITC, OX35), CD8a (PE-Cy7, OX8), CCR7 (APC, 4B12; all eBioscience) and CD45RC (PE, OX22; BD). Prior to CCR7 staining the FcgIIIR/FcgIIR was blocked with purified CD16/32 (eBioscience). Fluorescence intensity was analyzed on a FACS Calibur (BD) and data analysis was performed using CellQuestPro software. Graft survival was assessed by Kaplan�CMeier log rank survival analysis. Values are expressed as mean?��?standard deviation (SD) or standard error of mean (SEM, correolide C plasma levels, facs data). Analysis of variance (anova) was utilized to compare the differences between groups. http://www.selleckchem.com/products/ly2157299.html Correction of multiple comparisons was performed according to Bonferroni. A P-value of