Mitochondrial Isolation: A Technique to Isolate Mitochondria from Human Ovarian Cancer Tissue Sample
Transcrição
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.
