Mitochondria from Cultured Cells
HEPES 7.2 5mM
Dissolve in 900ml dd/MilliQ H2
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 4o
5% BSA in dd/MilliQ H2
Sterile filter (with 0.22 m m filter) and store at 4o
Use a refrigerated centrifuge at all centrifugation steps. Keep all the pellet and supernatant fractions on ice during isolation.
- 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.
- 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.
- Disrupt the cells in a Dounce homogenizer with 10-20 passes of a pestle.
- Add 10ml of isolation buffer and centrifuge at 625 g for 5min. collect the supernatant with a pipette.
- Repeat the centrifugation of the supernatant two more times.
- Collect the mitochondrial pellet from the final supernatant by centrifuging at 10000 g for 20min at 4oC.
- 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.
- 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 -80oC.
Trounce IA et al
., (1996) Methods in Enzymology 264: 484-509