Colorimetric assay

The activity of the caspase-3 was measured using a colorimetric assay (Elabscience). Centrifugation at 1×10³ g for 10 min was used to collect cells that had been exposed to six different concentrations of Ca²⁺ for 15 and 60 min. Lysis buffer was used to lyse the pelleted cells. Then, the cell lysates were incubated on ice for 10 min and were centrifuged at 14×10³ g for 1 min. Following the centrifugation, supernatants were transferred to new tubes. For measuring caspase-3 enzyme activity, 100 µl of the samples were added to the wells, after the incubation period 100 µl Biotinylated Detection Antibody working solution, 100 µl HRP conjugate working solution, 90 µl Substrate Reagent and 50 µl Stop Solution applied to each well respectively and incubated for 2 h at 37°C in CO₂ incubator. Absorbances of the samples were read under 450 nm via the ELISA reader (Biotek).

Caspase-3 enzyme activity was determined using a colorimetric assay (Elabscience). The cells that had been treated with IM, Das, LPS and TNFα and/or Verb for 48 h were collected by centrifugation at 1,000 g for 10 min. Pelleted cells were treated with 100 µl of cold lysis buffer to obtain the cell lysate. Then, the cell lysates were incubated on ice for 10 min and were centrifuged at 14,000 g for 1 min. Following the centrifugation, supernatants were transferred to new microcentrifuge tubes. For measuring caspase-3 enzyme activity, 100 µl of the samples were added to the wells, after the incubation period 100 µl Biotinylated Detection Ab working solution, 100 µl HRP conjugate working solution, 90 µl Substrate Reagent and 50 µl Stop Solution applied to each well respectively. Absorbances of the samples were read under 450 nm wavelength of light via a microplate reader.