The cultures were centrifuged for 5 minutes at 400 x g . Cell pellets were resuspended in a trypan blue solution and counted using a hemocytometer. The plates were returned to 37 C for the duration of the assay. The contents of culture wells were centrifuged at 450 x g for 5 minutes, and 1ml of supernatant was removed and stored frozen at 20 C until analyzed. Relative LDH values were determined at A 490 after a 30-minute enzymatic development.
Data represent percent of total lysis control.
After one hour, the contents of the well were mixed and 100 l aliquots were removed and transferred to a 96 well flat-bottom tissue culture plate. Absorbance at 490nm was recorded. Data represent percent of zero-hour control. Cells were harvested from T-25 flasks by centrifugation at 450 x g for 7 minutes. Samples containing 30 g of protein were used to determine CPP32 activity.