Seed HeLa cells in 15 cm dishes and grow until confluence. Treat the cells with DMSO or the mitophagy-inducing cocktail Oligomycin/Antimycin A (O/A) if needed.

Collect HeLa cells by trypsinization and resuspension in DMEM medium. Centrifuge the cells at 300x g,4°C.

Resuspend the cell pellet in 1mL of ice-cold PBS (1x) and spin again for 300x g,4°C.

Remove the PBS and resuspend the cell pellet in 1mL mitochondrial isolation buffer, which was cooled to 4°C.

To lyse cells without damaging the mitochondria, pipet the 1mL cell suspension up and down (15-20x) with a 26.5G needle.

Spin the suspension down at 600x g,4°C.

Keep the supernatant and centrifugation 600x g,4°C.

After two spins at 600x g,0h 0m 0s, subject the supernatant to 7000x g,4°C.

The pellet contains the mitochondria, so remove the supernatant and resuspend the pellet in 1mL mitochondrial isolation buffer.

Then, centrifugation 7000x g,4°C.

After two spins at 7000x g,0h 0m 0s, subject the resuspended pellet to 10000x g,4°C.

The pellet contains the mitochondria, so remove the supernatant and resuspend the pellet in 1mL mitochondrial isolation buffer.

Then, centrifugation 10000x g,4°C.

After two spins at 10000x g,0h 0m 0s, the pellet can be resuspended in RIPA lysis buffer or used for further procedures such as mitochondrial import assays, proteinase K protection assays, etc.