Isolation of Mitochondria from Hard Tissues (Skeletal or Heart Muscle) using the FOCUS™ Mitochondria Kit
Use a fresh tissue sample (obtained within one hour of sacrifice) kept on ice.
Do not freeze.
Weigh approximately 50-100mg tissues.
On a cooled glass plate, with the aid of a scalpel, mince the tissue into very small pieces.
Suspend the sample with 8 volumes of 1X SubCell Buffer-II containing 0.25mg/ml trypsin in a 2ml centrifuge tube.
Incubate on ice for 3 minutes and then spin down the tissue for a few seconds in the centrifuge.
Remove the supernatant by aspiration and add 8 volumes of 1X SubCell Buffer-II containing 0.25mg/ml Trypsin.
Incubate on ice for 20 minutes.
Add BSA Solution to a final concentration of 10mg/ml and mix.
Spin down the tissue at 1,000 x g for 5-10 seconds in the centrifuge.
Remove the supernatant by aspiration.
Wash the pellet with 8 volumes of 1X SubCell Buffer-II without Trypsin, and spin down the tissue for a few seconds in the centrifuge.
Remove the supernatant by aspiration and add 8 volumes of the 1X SubCell BufferII without Trypsin.
Transfer the suspension to an ice-cold Dounce tissue homogenizer and using a loose-fitting pestle disaggregate the tissue with 5-15 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.
Stand on ice for 2 minutes.
Transfer the homogenate to a centrifuge tube and leave large chunks of tissue in the homogenizer to be discarded.
Centrifuge the lysate at 700 x g for 5 minutes to pellet nuclei.
Transfer the supernatant to a new tube.
Centrifuge it at 12,000xg for 10 minutes and remove the supernatant.
The pellet contains mitochondria.
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.
