the manufacturer 39 s instructions miltenyi biotech usa auburn calif cells were then labeled for 20 minutes with 2 u03bcm 5 6 carboxyfluorescein diacetate n succinimidyl ester cfse sigma aldrich in hank 39 s balanced salt solution hbss mediatech herndon va cells were plated at a concentration of 2 5 u00d7 10 6 ml in dulbecco 39 s modified eagle 39 s medium dmem mediatech supplemented with 15 heat inactivated fetal calf serum sigma aldrich st louis mo penicillin 100 u ml streptomycin 100 u03bcg ml 2 mm l glutamine 10 mm hepes 0 1 mm nonessential amino acids 1 mm sodium pyruvate mediatech complete dmem cells were treated with 1 u03bcg ml anti cd3 antibody 2c11 30 and 1 u03bcg ml anti cd28 antibody 37 51 gross et al j immunol 149 380 1992 cells were cultured at 37 c 5 co 2 for the indicated time for studies of atm u2212 u2212 t cell proliferation in response to mitogens purified t cells were cultured with 2 u03bcg ml concanavalin a or 10 ng ml pma and 1 u03bcg m ionomycin stimulation of t cells in the presence of caffeine splenocytes were harvested from c57bl 6 mice jackson laboratories and re suspended at 3 u00d7 10 6 cells ml in complete dmem with 1 u03bcg ml anti cd3 and cd28 and various concentrations of caffeine acros organics n j or the same volume of water some cultures were in addition treated with 2 mm n acetyl cysteine sigma aldrich stimulation of t cells in the presence of ku 55933 t cells were purified.