Cytochrome c Releasing Apoptosis Assay Kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
- Cells were pre treated with artonin E at different time points	- Cells were Dounce-homogenized before centrifuging at 7,000×g for 10 min at 4°C.

Upstream tips
- Cells were pre treated with artonin E at different time points
Protocol tips
- Cells were Dounce-homogenized before centrifuging at 7,000×g for 10 min at 4°C.

Publication protocol

Cytochrome c releasing apoptosis assay kit (ab65311) was purchased from abcam® (Cambridge, UK). The assay was conducted according to the manufacturer’s instruction. Briefly, SKOV-3 cells were induced with artonin E at different time points. The induced and un-induced cells were collected and centrifuged at 6,00×g for 5 minutes at 4°C. The cells were washed twice with 10 mL of ice-cold PBS, centrifuged and collected the pellet. The fresh pellets were then re-suspended with 1.0 mL of 1X cytosol extraction buffer mix containing DTT and Protease Inhibitor and incubated on ice for 10 minutes. Cells were then Dounce-homogenized before centrifuging at 7,000×g for 10 min at 4°C. The supernatant were collected and saved as cytosolic fractions. The remaining pellets were resuspended in 0.1 mL mitochondrial extraction buffer, vortexed for 10 seconds and saved as mitochondrial fractions. Both cytosolic and mitochondrial fractions were applied for protein analysis and proceeded with standard Western blot procedure.

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