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 .
