Daan Pieren

67 Compromised DNA repair promotes the accumulation of regulatory T cells Since Ercc1 -/ Δ 7 mice are smaller, food was administered within the cages and water bottles with long nozzles were used from ~2 weeks of age. Wild-type F1 Ercc1 +/+ littermates at the indicated ages were used as controls. Male and female Ercc1 -/ Δ 7 and Ercc1 +/+ mice were maintained at the animal facilities of the Erasmus Medical Centre (Rotterdam, Zuid-Holland, The Netherlands). Distribution of males and females per group; n=3 males and n=3 females in the group of Ercc1 +/+ mice, n=3 males and n=4 females in the group of Ercc1 -/ Δ 7 mice, and n=3 males and n=3 females in the group of Ercc1 -/ Δ 7 mice fed with eRapa. Dietary treatment with rapamycin Diets were based on AIN93G, using 2.3 g/kg choline chloride instead of choline bitartrate (Research Diet Services, Wijk bij Duurstede, the Netherlands). Microencapsulated rapamycin (eRapa) and empty microcapsules (Eudragit S100) were obtained from Southwestern Research Institute (San Antonio, TX, USA). 42mg eRapa, containing 10% Rapamycin, was added per kg AIN93G food mix, resulting in a 42ppm rapamycin supplemented diet. For the control diet, 38mg empty microcapsules were added per kg AIN93G food mix. The diets were processed into pellets which were radiated with 9 kGy (Isotron, Ede, the Netherlands). Supplemented eRapa and control diets were supplied ad libitum to the mice at 8 weeks of age for the remainer of their life. Ethics Animal studies were approved by the Animal Ethical Committee of the National Institute for Public Health and the Environment (DEC no. 201400042) or Erasmus Medical Centre (DEC no. 139-12-13). All procedures were carried out in accordance with Dutch national legislation. Preparation of single cell suspensions and proliferation labeling Spleens of all mice were homogenized through a cell strainer to prepare single cell suspensions and red blood cells were lysed on ice with ACK lysis buffer (0.155M NH4Cl; 10mM KHCO3; 0.1mM Na2EDTA, pH 7.2-7.4). Subsequently, splenocytes were resuspended in PBS to 10*10 6 cells/mL and labeled with 0.5 µ M CellTrace TM Violet (Invitrogen, Carlsbad, CA, USA) in PBS per milliliter of splenocyte suspension for 20 minutes at 37°C to track T-cell proliferation. Cells were washed in ice-cold RPMI-1640 medium (GIBCO, Thermo Fisher Scientific, Waltham, MA, USA) containing 10% fetal calf serum (FCS) (Greiner Bio-One, Kremsmünster, Austria). 3