Determination of Caspase activity

Cells were grown to 7080% confluence in 12-well plates and were incubated for 12 hours and
24 h with various concentrations of fraction with 10% FBS-DMEM. The activities of caspase-3
and Caspase-8 and Caspase-9 were determined chromogenic by cleavage of substrates: AcDEVD-7-amino-4-methylcoumarin pNA, Ac-IETD-pNa and Ac-LEHD-pNA respectively, (Short
caspase specific peptides) according to described methods (Kohler et al., 2002). Approximately
5x104 cells were used. The Sample concentration was used from range of 100-400 ng/well. The
apoptotic cells were estimated by elevated level of intracellular compared with and without
compound treated cells. Cleavage of the chromogenic peptide substrates was monitored by pNA
release in a 405 nm scan II plate reader (Biorad, USA). Chromogenic units were converted to
percentage by comparing the untreated control cells generated free pNA. Quantification of
caspase activity was calculated as fold increase over control samples. Positive control
Camptothecin (CPT) used as 1 g/ml concentration.
A405 of drug-treated sample
Calculation =

X 100
A405 of control