Mitochondria from Cultured Cells

Isolation Buffer:

HEPES 7.2 5mM

Mannitol 210mM

Sucrose 70mM

EGTA 1mM

BSA 0.5%

A

1.19g HEPES

38.26g Mannitol

23.96g Sucrose

Dissolve in 900ml dd/MilliQ H₂O.

Add 0.38g EGTA slowly while stirring to solubilize it.

Adjust pH to 7.2 with 1M KOH (K+ stimulates respiration)

Make it up to 1Lt with dd/MilliQ H2O.

Sterile filter with 0.22 m m filter

Store at 4^oC

B

5% BSA in dd/MilliQ H₂O.

Sterile filter (with 0.22 m m filter) and store at 4^oC

Procedure:

Use a refrigerated centrifuge at all centrifugation steps. Keep all the pellet and supernatant fractions on ice during isolation.

1. Wash a 1-2x10⁹ cell pellet with 50ml cold isolation buffer and pellet at 450 g for 3min. Discard the supernatant and weigh the pellt.
2. Add 5ml more of isolation buffer and centrifuge at 3000 g for 5min. Discard the buffer and suspend the cell pellet in 5 times its weight of isolation buffer.
3. Disrupt the cells in a Dounce homogenizer with 10-20 passes of a pestle.
4. Add 10ml of isolation buffer and centrifuge at 625 g for 5min. collect the supernatant with a pipette.
5. Repeat the centrifugation of the supernatant two more times.
6. Collect the mitochondrial pellet from the final supernatant by centrifuging at 10000 g for 20min at 4^oC.
7. Gently rinse the pellet with a few ml of the buffer, dissolve in 25ml of isolation buffer and pellet it by centrifuging again at 10000g for 20min.
8. Collect the pellet by dissolving in a few ml of isolation buffer and re-pelleting in a micro-fuge tube at maximum speed for 10min in a bench-top. The dry pellet can be stored below -80^oC.

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