This article details the isolation of mitochondria from cancerous tissues, highlighting the structural and functional changes during cancer progression. The methodology involves a series of centrifugation steps to separate and purify mitochondria from other cellular components.
Mitochondria, the cytoplasmic organelles known as the cell's powerhouses, undergo drastic structural and functional changes during cancer progression.
To isolate mitochondria, take clean cancerous tissue in a glass dish and mince it into small pieces. Transfer the pieces into a tube containing mitochondrial isolation buffer. Homogenize the sample to disrupt the cells and release their contents, including the organelles.
Centrifuge the mixture at a low speed. This pelletizes the denser components like nuclei, tissue debris, and any intact cells, leaving less dense components like mitochondria, microsomes, peroxisomes, and lysosomes in the supernatant. Collect the supernatant and centrifuge at high speed. Microsomes, being relatively lighter, remain in the supernatant, while the crude mitochondria-containing fraction pelletizes at the bottom.
Remove the supernatant and resuspend the pellet in a suitable density gradient medium. Set up a discontinuous density gradient by layering the crude mitochondrial fraction within different density media, arranging them in decreasing order of densities from bottom to top. Centrifuge at a very high speed. Organelles separate within the tube to adopt an equilibrium position based on their relative densities. Collect the mitochondrial fraction formed at the interface between the density media, containing peroxisomes and lysosomes.
To begin this procedure, prepare 250 milliliters of the mitochondrial isolation buffer as outlined in the text protocol. Place approximately 1.5 grams of ovarian cancer tissues in a clean glass dish. Add 2 milliliters of pre-chilled mitochondrial isolation buffer to lightly wash the blood from the tissue surface. Repeat this wash three times.
Then, use clean ophthalmic scissors to fully mince the tissue into pieces that are approximately one cubic millimeter and transfer the minced tissues into a 50-milliliter centrifuge tube. Add 13.5 milliliters of mitochondrial isolation buffer containing nagarse at a concentration of 0.2 milligrams per milliliter. Use an electric homogenizer to homogenize the minced tissues for two minutes at 4 degrees Celsius.
After this, add another 3 milliliters of mitochondrial isolation buffer to the tissue homogenates and mix them well by pipetting. Centrifuge the prepared tissue homogenate at 1,300 x g and at 4 degrees Celsius for 10 minutes. Remove the pellet and keep the supernatant.
Next, centrifuge the supernatant at 10,000 x g and at 4 degrees Celsius for 10 minutes. Remove the microsome-containing supernatant and keep the pellet. Add 2 milliliters of mitochondrial isolation buffer and resuspend the pellet thoroughly by light pipetting.
Centrifuge this pellet suspension at 7,000 x g and at 4 degrees Celsius for 10 minutes. Discard the supernatant and keep the pellet, which contains the crude mitochondria. Add 12 milliliters of 25% density gradient medium to resuspend the extracted crude mitochondria. Make a discontinuous density gradient containing the resuspended crude mitochondria, as outlined in the text protocol, and centrifuge it at 52,000 x g and at 4 degrees Celsius for 90 minutes.
Use a long and blunt syringe to collect the purified mitochondria at the interface between the 25% and 30% gradient mediums and transfer this to a clean tube. Add mitochondrial isolation buffer to the collected mitochondria to dilute it to a three-fold volume. Centrifuge at 15,000 x g and at 4 degrees Celsius for 20 minutes. Discard the supernatant and keep the pellet. Collect the final pellet, which contains the purified mitochondria, and store it at minus 20 degrees Celsius.
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