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. Reagents were allowed to equilibrate to 30 C before the CPP32 substrate was added. Changes in gross cell morphology were observed microscopically during the first 2 hours of anisomycin treatment. The parallel release of LDH and staining with the vital dye trypan blue is expected because these two methods are both indicators of damage to the cell membrane.