(B) Data from five independent experiments as in (A) above showing proportions of subdiploid cells from ANCD4 and YNCD4 cultures. this point 98% of the cells were viable as checked by Trypan blue exclusion and 95% by annexin V. In vitro T cell stimulation assays ANCD4 cells and naive CD4 cells from young mice (YNCD4 cells) or purified CD4+ EM cells from Yg and Ad mice were stimulated with plate-coated anti-CD3 and anti-CD28 mAbs (1 and 5 g ml?1 respectively; BD Biosciences) or bead-coated anti-CD3 and anti-CD28 mAbs (Dynal via Invitrogen; used at a 1?:?1 cells?:?beads ratio] and were compared with unstimulated cells. Under these activating conditions, the first division of ANCD4 and YNCD4 cells was observed to take place after 36 h (data not shown). Where indicated, 3-methyl adenine (3-MA; Sigma Aldrich, Bangalore, India) was added at a final concentration of 1 1 mM. T cell blasts were prepared as described earlier (25). Briefly, ANCD4 and YNCD4 cells were stimulated with anti-CD3 and anti-CD28 mAbs for 48 h, and cells were cultured in the presence of 5 U ml?1 of IL-2 for a further 48 h. At the end of 96 h, viable cells were obtained by density-gradient centrifugation. These 96-h T cell blasts were subjected Daurisoline to further experiments. For measuring proliferation of purified EM cells or 96-h T cell blasts, 1 105 T cells were cultured in anti-CD3-coated 96-well plates and were pulsed at 48 h for measurement of Daurisoline [3H]thymidine incorporation. Data are expressed as mean SEM of triplicate cultures. For antigen-specific recall assays, draining lymph node cells were stimulated with 100 g ml?1 of mOA. Levels of secreted IFN-gamma were measured from culture supernatants collected at 48 h by ELISA, essentially following manufacturer’s recommendations (BD Biosciences). Standards were run in parallel to enable calculation of concentrations of IFN-gamma. Flow cytometry For flow cytometry, cells were stained with primary and secondary antibodies as appropriate on ice, washed and analysed immediately. For cell cycle analysis, cells were harvested, fixed and permeabilized by 70% ethanol and propidium iodide (PI) was added at a final concentration of 10 g ml?1. For scoring the cells undergoing loss of mitochondrial potential, cells were stained with 3,3-dihexyloxacarbocyanine iodide (DiOC6; Molecular Probes, Invitrogen) in serum-free medium and PI was added to a final concentration of 1 1 g ml?1. Cells were analysed immediately. Stained samples were run either on FACSort, LSR I or FACSaria flow cytometers (Becton and Dickinson, San Jose, CA, USA), and data were analysed using FlowJo software (Tree Star, San Carlos, CA, USA). Comet assay ANCD4 and YNCD4 cells were activated for 12 or 24 h and harvested, and dead cells were removed by density-gradient centrifugation so that viability of the cells assayed was 99% by Trypan blue exclusion test before assay. The cell suspension was then mixed with low-melting agarose and poured on a slide. The relative proportions of DNA with breaks in individual cells were measured following the alkaline KIAA1823 comet assay procedure (26). Alternatively, ANCD4 and YNCD4 were exposed to 3 Gy gamma-radiation using Co60 as a source and the extent of damaged DNA was scored as above after 12 h. Pictures were taken under a Daurisoline fluorescence microscope (Olympus America Inc., Center Valley, PA, USA) and between 30 and 60 cells were analysed per sample by CometScore software (TriTek Corp., Virginia, VA, USA) (26). Confocal microscopy and quantitation Stimulated or unstimulated ANCD4 and YNCD4 cells were harvested at indicated times, fixed and permeabilized using 0.5% saponin and 4% paraformaldehyde (both from Sigma Aldrich) as a modification of a published method (27). Cells were stained with rabbitCanti-LC3 antibody followed by goatCanti-rabbit F[ab]2-Alexa fluor 488. Cells were also stained with anti-CD4 and DAPI to visualize the cell surface and nucleus, respectively. Cells were analysed under a Leica TCS SP2 laser confocal microscope (Leica Microsystems,.