for 72 hr both floating and attached cells were collected by centrifugation cells at a density of 1 5 u00d7 10 6 cells ml were washed with pbs and then re suspended in chilled cell lysis buffer supplied in the caspase 3 colourimetric assay kit calbiochem uk cells were incubated on ice for 10 min and then centrifuged for 1 min at 10 000 g the cytosolic fraction supernatant was collected into a fresh tube and 50 u03bcl 2 mg ml of each protein sample was transferred to a 96 well plate and diluted with lysis buffer a 50 u03bcl aliquot of u00d7 2 reaction buffer containing 10 mm dtt was added followed by 5 u03bcl of 4 mm devd pna substrate to give a final concentration of 200 u03bcm the plate was then incubated at 37 u00b0 c for 1 2 h od was determined at 400 nm using an anthos ht ii microtitreplate reader the fold increase in caspase 3 and 9 activities was determined by comparing results to that obtained with un induced control a2780 p and a2780 cisr cells were grown in t75 flasks to 70 confluency and exposed to test agents at concentrations equivalent to the ic 50 and ic 75 for 72 hr both floating and attached cells were collected by centrifugation washed twice in ice.