1 × 105 cells/mL were treated with IC50 of CLG and incubated for 24 and 48 h while untreated cells acted as control. The cells were lysed by the addition of 50 μL of chilled Cell Lysis Buffer and incubated on ice for 10 min. The resulting cell lysate was centrifuged for 1 min at 10,000 ×g, and the supernatant was collected. Fifty microliters of 2X Reaction Buffer (containing 10 mM DTT) was added to each sample. Then 5 μL of DEVD-pNA (caspase-3 substrate) was added and incubated in the dark at 37°C for 1 h. At the end of the incubation period, the samples were read at 405 nm in a microplate reader (TECAN, SunriseTM, Männedorf, Switzerland). Data was presented as optical density (405 nm; ).
1 × 105 cells/mL were treated with IC50 of CLG and incubated for 24 and 48 h while untreated cells acted as control. The cells were lysed by the addition of 50 μL of chilled Cell Lysis Buffer and incubated on ice for 10 min. The resulting cell lysate was centrifuged for 1 min at 10,000 ×g, and the supernatant was collected. Fifty microliters of 2X Reaction Buffer (containing 10 mM DTT) was added to each sample. Then 5 μL of DEVD-pNA (caspase-3 substrate) was added and incubated in the dark at 37°C for 1 h. At the end of the incubation period, the samples were read at 405 nm in a microplate reader (TECAN, SunriseTM, Männedorf, Switzerland). Data was presented as optical density (405 nm; ).
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