Isolation of Mitochondria from Soft Tissues (Liver or Brain) using the FOCUS™ Mitochondria Kit
OPTIONAL: Delipidated BSA can be added to 1X SubCell Buffer-II to the concentration of 2mg/ml for removing free fatty acids prior processing.
An appropriate amount of protease inhibitor cocktail also can be added to the 1X SubCell Buffer-II just before use.
Use a fresh tissue sample (obtained within one hour of sacrifice) kept on ice.
Do not freeze.
Weigh approximately 50-100mg tissue.
On a cooled glass plate, with the aid of a scalpel, mince the tissue into very small pieces.
Perform this step on ice.
Transfer the minced tissue to an ice-cold Dounce tissue homogenizer.
Stand on ice for 2 minutes.
Transfer the homogenate to a centrifuge tube and leave large chunks of tissue fragments in the homogenizer to be discarded.
Centrifuge the lysate at 700x g for 5 minutes to pellet the nuclei.
Carefully transfer the supernatant into a new tube.
Centrifuge supernatant at 12,000x g for 10 minutes.
Remove the supernatant and resuspend the pellet in 10 volumes of 1X SubCell Buffer-II without BSA.
(Centrifuge as in steps 9-10.)
Transfer the homogenate to a centrifuge tube and leave large chunks of tissue fragments in the homogenizer to be discarded.
Suspend the mitochondrial pellet in Working Mitochondria Storage Buffer (approximately 50μl for pellet from ~100mg tissue) and keep the suspension on ice before downstream processing.
The suspension may be stored on ice for up to 48 hours.
Add 10 volumes of 1X SubCell Buffer-II and using a loose-fitting pestle disaggregate the tissue with 5-10 strokes or until the tissue sample is completely homogenized.
Using a tight-fitting pestle, release the nuclei with 8-10 strokes.
Do not twist the pestle as nuclei shearing may occur.
(Centrifuge as in steps 9-10.)
Centrifuge the lysate at 700x g for 5 minutes to pellet the nuclei.
Carefully transfer the supernatant into a new tube.
Centrifuge supernatant at 12,000x g for 10 minutes.
Remove the supernatant.
The pellet contains mitochondria.
