H.T.T. a non-monotonic response pattern as determined by the TRAP-ELISA assay. Under long-term BPA exposure, significant telomere length shortening, reduction in mitochondrial DNA copy number, cell proliferation and IFN- as well as hTERT protein suppression could be observed in CD8?+?lymphocytes, as analysed by qRT-PCR, flow cytometry and western blot analysis. This study extends our previous in vitro findings that low-dose BPA has potential negative effects on healthy human cytotoxic T cell response. These results might merit some Deoxycholic acid special attention to further investigate chronic BPA exposure in the context of adaptive immune response dysfunction and early onset of cancer in man. Telomerase PCR ELISA Kit, from Roche (Mannheim, Germany) as described before62. In brief, 0.5??106 cells were lysed according to the manufacturers protocol. An equal amount of protein as determined by the Bradford method63 was added to the reaction mixture to a final volume of 50?l. The reaction was performed in an Eppendorf Mastercycler Nexus Thermal Cycler (Thermo Fisher Scientific GmbH, Dreieich, Germany) following these steps: 25?C for 20?min, then denatured at 94?C for 5?min, 30 cycles (94?C for 30?s, 50?C for 30?s and 72?C for 90?s), followed by elongation at 72?C, 10?min. A heat-treated sample (95?C for 5?min) was used as negative control. Subsequently, 5?l of the PCR product were denatured and hybridised with adeoxigenin-labelled telomeric repeat probe. Absorbance was measured at 450?nm (reference 690?nm) using a multiplate reader from Tecan (Tecan Group Ltd, Crailsheim, Germany). RNA isolation Total RNA was isolated from 4??106 CD8?+?T cells Deoxycholic acid by using the RNeasy mini Isolation kit from Qiagen (Hilden, Germany) followed by a purification step using the RNase-free DNase kit from Qiagen (Hilden, Germany) according to the manufacturers instructions42. RNA quality and quantity were assessed using a NanoDrop ND-1000 spectrophotometer (Thermo Scientific, Freiburg, Germany). Human DNA repair RT-PCR array 1?g RNA was taken for cDNA synthesis using the RT2 PCR array First Strand Synthesis Kit (Qiagen, Hilden, Germany). The qPCR of RT2 Profiler PCR Array Human DNA Repair (cat no: PAHS-042Z from Qiagen, Hilden, Germany) was performed using a CFX96 Touch Real-Time PCR Detection System (Bio-Rad, Mchen, Germany). The PCR was carried out at 95?C for 15?min, followed by 40 PCR cycles (95?C for 10?s, 60?C for 15?s, rampe rate 1?C/s) and a final extension for 5?min at 72?C, followed by a standard melting curve analysis. The web-based automated RT2 Profiler PCR Array Data Analysis from the manufacturer was used to analyse the data (https://geneglobe.qiagen.com/us/analyze/). Deoxycholic acid DNA isolation Genomic DNA was isolated from 1.5??106 CD8?+?T cells using the FlexiGene DNA Kit from Qiagen (Hilden, TSPAN33 Germany) according the manufacturer’s instructions. DNA purity and quantity were determined using a NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific GmbH, Dreieich, Germany). Telomere length quantification Full telomere length Deoxycholic acid was quantified based on the method of O’Callaghan and Fenech64 using the 36B4 gene as reference. Each sample contained 20?ng of purified DNA, 2??Maxima SYBR Green qPCR master mix (Thermo Fisher Scientific GmbH, Dreieich, Germany), forward and reverse primers for the telomere or 36B4 gene. Each sample was run in triplicate using a CFX96 Touch Real-Time PCR detection System (Bio-Rad, Munich, Germany). The cycling conditions were set at 95?C for 10?min, followed by 40 PCR cycles (95?C for 15?s, 60?C for 1?min), and subsequent standard melting curve analysis. Data were analysed using the comparative CT method calculating the difference between the threshold cycle (CT) values of the target and reference gene of each sample and then comparing the resulting of the CT values between the different samples. Mitochondrial DNA copy number quantification Relative quantification of human mitochondrial DNA copy number was done using the Relative Human Mitochondrial DNA copy number quantification qPCR Assay kit (Provitro AG, Berlin, Germany) according to the manufacturers instructions. Briefly, 5?ng genomic DNA were mixed with 2??Maxima SYBR Green qPCR master mix ( Thermo Fisher Scientific GmbH, Dreieich, Germany) and mtDNA primer or the single copy reference (SCR) primer set. Quantitative PCR of 20?l total reaction volume was performed at 95?C for 10?min, followed by 40 PCR cycles (95?C for 29?s, 52?C for 20?s, 72?C for 45?s) and subsequent melting curve analysis. Data analysis was done as describe above. Flow cytometry Purity analysis of freshly isolated CD8?+?T cells (2??105) was done using an anti-CD8-APC mAb. Only T cell populations with a purity?>?90%, as determined by flow cytometry, were used for the experiments. For identifying co-stimulatory receptor expression, CD8?+?T cells (2??105) were stained with anti-CD8-APC mAb coupled with anti-CD28-PE and anti-CD27-PerCP mAbs for 30?min at 4?C. Staining for naive and memory cells was performed using anti-CD8-APC.