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2.4. Antibodies and Reagents Anti-mouse CD4 (clone RM4-5), anti-Helios (clone 22F6), anti-Foxp3 (clone FJK-16s), anti-ROR��t (clone AFKJS-9), anti-GATA-3 (clone TWAJ), anti-Tbet (clone eBio4B10), anti-IL-17A (clone eBio 17B7), anti-IFN-�� (clone XMG1.2), CD45RB (clone C363.16A), Rag IgG2a isotype control (clone eBR2a), anti-CD3 (clone 145-2C11), and anti-CD28 (clone 37.51) were purchased from eBioscience. Human TGF-��1 was purchased from R&D systems. Diphtheria toxin and collagenase type IV were purchased from Sigma. DNase I was purchased from Roche. 2.5. Flow Cytometry and Cell Culture For CD4 T cell differentiation, na?ve CD4 T cells (CD4+CD62LhiCD44loFoxp3(RFP)-) were isolated from 6-7-week-old ApcMin/+-Foxp3-IRES-RFP and littermate control mice. For inducible Treg transfer, splenic na?ve CD4+ T cells from 6-week-old ApcMin/+-Foxp3-IRES-RFP mice and their littermate controls (Foxp3-IRES-RFP) spleens were activated with anti-CD3 (2.5?��g/mL), IL-2 (100?U/mL), and 1.5?ng/mL of TGF-�� for 5 days. For LPMC (lamina propria mononuclear cell) preparation, 16-week-old ApcMin/+ mice and Treg-transferred ApcMin/+ mice were sacrificed, and the AZD6244 individual small intestines were processed as described with modification [19]. Briefly, small intestines were cut longitudinally and fecal contents were removed. Each small intestine was cut into 5?cm long pieces and extensively washed with PBS. Subsequently, small intestine pieces were incubated in shaking incubator with 5?mM EDTA-PBS solution. To digest these pieces, EDTA was removed and collagenase and DNase I solution was added in 5% FBS RPMI media for 30?min at 37��C. After Percoll gradient separation, cells were washed with ice-cold PBS twice, and lymphocytes were counted with trypan blue exclusion. Approximately 90% of cell viability was obtained. The yield of cells was presented in Supplementary Figure 1 in Supplementary Material available online at http://dx.doi.org/10.1155/2015/860106. For the stimulation of LPMC cells, PMA (100?ng/mL) and ionomycin (1?��M) were used. Brefeldin A (1?��g/mL) was added for the 4?h of culture in a total 6?hr of activation. After stimulation intracellular staining was performed as described in the manufacturer's protocol (eBioscience). A FACSCalibur (BD Biosciences) was used for flow cytometry and data were analyzed by FlowJo software (Treestar). 2.6. Real Time PCR Cells were harvested and washed with PBS and then suspended in Trizol. cDNA was synthesized with the BDsprint cDNA synthesis kit (Clontech) and used with SYBR Green (Molecular Probes). Real-time PCR was performed using a Real time Bioanalyzer (BioRad). Primers were synthesized by the Keck Biotechnology Institute (Yale University). We calculated relative gene expression by the ��CT method [20]. The mRNA expression was normalized against HPRT. Primer sequences are available upon requests.