Cytotoxicity Assays Protocol

Cell-mediated cytotoxicity was determined by using a standard microcytotoxicity assay. Briefly, target cells were pelleted and resuspended in 100 mCi of Na51Cr (DuPont NEN, Boston, MA) per 106 cells and incubated at 37oC in a humidified 5% CO2 incubator for 1 hour (hr). They were washed three times with media, resuspended to 5 x 104/ ml, and 0.1 ml was added to round-bottomed microtiter wells (Becton Dickinson & Company, Franklin Lakes, NJ). Varying numbers of effector cells were added in 0.1-ml volume to achieve the desired E/T ratios. For a spontaneous 51Cr-release control, 0.1 ml of media was substituted for effector cells. Maximum release was determined by adding 0.1 ml of 1% NP-40 (United States Biochemical) to the target cells. After 6 hr at 37 oC, the plates were centrifuged at 200 x g for 5 min, and 0.1 ml of supernatant was removed from each well and counted on a gamma counter (Model Cobra II, Packard Instrument Company). Data are presented as percent specific 51Cr-release = 100 x [(experimental cpm - spontaneous cpm) / (maximum release cpm - spontaneous cpm)].