Mitochondria isolation

Mitochondria isolation protocol for U2OS cells (adherent)

Homogenization buffer (adjusted to pH 7.4 at 4^oC):

• 10 mM HEPES
• 1 mM EDTA
• 210 mM mannitol
• 70 mM sucrose

Procedure:

1. Grow U2OS cells to confluency in 150mm Petri dishes.
2. Wash cells 3x with 15ml of ice-cold homogenization buffer.
3. Aspirate residual homogenization buffer from the last wash and scrape cells in 0.75ml of ice-cold homogenization buffer supplemented with 1× protease inhibitor cocktail (MilliporeSigma, Burlington, MA).
4. Transfer cell suspension to a glass Dounce homogenizer fitted with a tight pestle and lyse cells with 6-8 slow strokes. Assess cell lysis efficiency by mixing equal volumes of cell homogenate and 0.4% trypan blue solution and looking under a microscope. About 95% of the cells should be properly lysed without showing sign of nuclei damage.
5. Centrifuge the homogenate at ~1000 g for 5 min at 4^oC to pellet nuclei, unbroken cells, and large cellular debris.
6. Carefully collect the supernatant without disturbing the pellet and centrifuge at ~14,000 g for 10 min at 4°C.
7. Carefully discard the supernatant without disturbing the crude mitochondrial pellet and resuspend the pellet in homogenization buffer supplemented with 1× protease inhibitor cocktail. Use ~100ul of homogenization buffer for each 150mm plate of confluent U2OS cells.
8. Aliquot and snap freeze on liquid nitrogen.
9. Store aliquots at -80^oC until needed.

Mitochondria isolation protocol for K562 cells (suspension)

Swelling buffer (adjusted to pH 7.4 at 4^oC):

• 10 mM HEPES
• 1 mM EDTA

2x homogenization buffer (adjusted to pH 7.4 at 4^oC):

• 10 mM HEPES
• 1 mM EDTA
• 420 mM mannitol
• 140 mM sucrose

Homogenization buffer (adjusted to pH 7.4 at 4^oC):

• 10 mM HEPES
• 1 mM EDTA
• 210 mM mannitol
• 70 mM sucrose

Procedure:

1. Grow 75ml of K562 cells to confluency (~1 million cells per ml) in 150mm Petri dishes.
2. Centrifuge cells at 1000 g for 5 min at room temperature. Remove supernatant.
3. Resuspend cell pellet in 50ml of ice-cold homogenization buffer.
4. Centrifuge cells at 1000 g for 5 min at 4^oC. Remove supernatant.
5. Resuspend cell pellet in 2ml of swelling buffer supplemented with 1× protease inhibitor cocktail (MilliporeSigma, Burlington, MA).
6. Incubate on ice for 20min.
7. Transfer cell suspension to a glass Dounce homogenizer fitted with a tight pestle and lyse cells with 25 slow strokes. 
8. Immediately add 2ml of 2x homogenization buffer supplemented with 1× protease inhibitor cocktail (MilliporeSigma, Burlington, MA).
9. Centrifuge the homogenate at ~1000 g for 5 min at 4^oC to pellet nuclei, unbroken cells, and large cellular debris.
10. Carefully collect the supernatant without disturbing the pellet and centrifuge at ~14,000 g for 10 min at 4°C.
11. Carefully discard the supernatant without disturbing the crude mitochondrial pellet and resuspend the pellet in homogenization buffer supplemented with 1× protease inhibitor cocktail. Use ~400ul of homogenization buffer for 75ml of confluent K562 cells.
12. Aliquot and snap freeze on liquid nitrogen.
13. Store aliquots at -80^oC until needed.

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