Isolation of Mitochondria from Animal Cells using the FOCUS™ Mitochondria Kit
OPTIONAL: Add appropriate protease inhibitor cocktail (e.g. G-Biosciences’ Protease Arrest, Cat# 786-108) to SubCell Buffer-I just before use.
Use fresh cells only.
Pellet the harvested cells by centrifugation at ~800 x g for 1 minute.
Carefully remove and discard the supernatant.
Add 500µl of ice cold SubCell Buffer-I.
Gently vortex to suspend the cells and incubate on ice for 10 minutes.
Perform this lysis step on ice.
Using a narrow opening (20 gauge) syringe needle, gently pull the suspension up and down 10-30 times.
(Alternatively, use Dounce homogenizer as described in the annotation below.)
Add 250µl 3X SubCell Buffer-II (350µl if Dounce homogenizer is used) and mix by inverting.
This generates a 1X final concentration of SubCell Buffer-II.
Centrifuge the tube at 700x g for 10 minutes to pellet the nuclei.
Transfer the supernatant to a new tube.
Centrifuge supernatant at 12,000x g for 15 minutes.
The pellet contains mitochondria.
Transfer the supernatant (cytosol fraction) to a new tube.
Add 500µl 1X SubCell Buffer-II to the pellet, and centrifuge again at 12,000 x g for 5 minutes.
Discard the supernatant.
Suspend the mitochondrial pellet in 50-100µl. 
Working Mitochondria Storage Buffer and keep the suspension on ice before downstream processing.
The suspension may be stored on ice for up to 48 hours.
