How do I isolate mitochondria from cell culture?

Question

Accepted Answer

Grow cells to 85-95% confluence in three large tissue culture plates (500 cm2 each)
Remove all growth medium once the cells reach the desired confluence.
Wash the cell layer twice with 30 ml of cold STE (buffer) and remove the buffer each time.
Scrape cells off the plate using a razor blade. 
Resuspend the scraped cells in 5 ml cold STE, transferring to a 50ml centrifuge tube. Repeat this process twice. 
Repeat steps 2-4 for all plates, pooling cells into separate centrifuge tubes (one tube per plate). 
Centrifuge the tubes at 2000 rpm for 10 minutes at 4˚C. 
Carefully remove the supernatant, keeping the slightly diffuse cell pellet.
Resuspend cell pellets in 3.5 ml cold STE with protease inhibitor and 0.5% BSA. Transfer to a cold 7ml glass-teflon homogenizer. Wash tubes with 1.5 ml STE with 0.5% BSA and transfer to the homogenizer. 
 Homogenize cells with 10 slow passes of the "tight" plunger. Transfer the homogenate to a 50 ml centrifuge tube, adding ice cold STE if needed. 
Centrifuge the homogenate at 3000 rpm for 3 min at 4˚C (1 tube). 
Strain the supernatant through wet muslin and spin at 10000 rpm for 11 min at 4˚C (2 tubes). 
Decant the supernatant, wipe tube walls. 
Resuspend the mitochondrial pellet in ice cold STE and transfer to a 15ml centrifuge tube. Add ice cold STE and spin at 11,600 G for 10 minutes. 
Re-suspend the mitochondrial pellet in residual supernatant using a cold test tube as a pestle. 
Keep the isolated mitochondria on ice for further use.

How do I isolate mitochondria from cell culture?

Posted February 9, 2024