Glomax multiplus luminometer

CTL were washed once with PBS (pH7.4) and resuspended in growth medium without phenol red and supplemented with 20mM HEPES and 10% FBS. Cells were plated into 96-well, round-bottom, white plates (Corning) in replicate wells at 50 μl per well. Biosensor-expressing EL4 targets were harvested and washed with PBS, followed by incubation with 2% (v/v in phenol red free growth media) biosensor substrate at 37°C, 5% CO2 for 30 minutes and added to effector wells, 50μl per well containing 5,000–20,000 cells. The total volume per well for the assay was 100μl. Following centrifugation at 200 g for 5 minutes, the plate was incubated at 37°C in GloMax Multi Plus luminometer (Promega) and luminescence was measured kinetically every 3 minutes with integration time of 0.5 seconds. Each data point represents the mean of four replicate wells. A similar protocol was used for testing NK function against YAC target cells. For the re-directed killing assay, P815 cells were pre-incubated with OKT3 at 5 μg/ml for 30 minutes and then co-incubated with human T cells to trigger the biosensor.