I am interested in analyzing the effects of some immunotherapy on the number of mitochondria in cancer cells as well as in immune cells. I would like also to isolate mitochondria to use them for further analysis far from other cellular compartments.
Dear Dr. Mohamed Labib Salem
Mitochondria are highly dynamic organelles undergoing coordinated cycles of fission and fusion, referred as ‘mitochondrial dynamics’, to maintain their shape, distribution and size. The number of mitochondria in a cell depends on cell activity and type. For instance, mitochondria multiply when the energy needs of a cell increase. Therefore, power-hungry cells have more mitochondria than cells with lower energy needs.
Mitochondria can take up as much as 25% of the cell volume. Cells contain approximately 1000 to 2500 mitochondria. It would be difficult to count the exact number of mitochondria in the cells. Even if you try to isolate mitochondria from cells, the count will not reflect the exact number present in the cell because you would lose quite a number of them during the isolation process.
Attached below are a few articles for your reference.
Article On the problem of counting and sizing mitochondria: A genera...
Article Mitochondrial dynamics: Overview of molecular mechanisms
Article Mitochondrial dynamics: Shaping and remodeling an organelle network
Article Mitochondria—Fundamental to Life and Health
So, instead of the count I would suggest you work on the mitochondrial mass of cells which is evaluated by means of one of the following methods:
1. the biochemical method based on the assay of the matrix specific enzyme CS,
2. the genetic method based on the measurement of the mtDNA copy number, or
3. by evaluating the mass of typical mitochondrial proteins as translocase outer membrane (TOM) subunits through immunoblotting.
For more information regarding the above (mitochondrial mass) you may want to refer to the article attached below.
Article Mitochondrial Mass Assessment in a Selected Cell Line under ...
The second part of your question.
Isolation of mitochondria from single cell suspension.
1. Having obtained single cell suspension, wash them with PBS once and centrifuge at 300 × g for 3 min
2. Discard the supernatant and resuspend with 5 volumes of extraction buffer.
Extraction buffer recipe:
20 mM HEPES-KOH, pH 7.5,
0.25 M sucrose,
10 mM KCl,
1.5 mM MgCl2,
1 mM EDTA,
1 mM EGTA,
1 mM dithiothreitol,
0.1 mM PMSF
3. The cellular suspension is homogenized with a Teflon-glass homogenizer with 10–20 up-and-down passes of the pestle. Use of a teflon-glass homogenizer is recommended as it can lead to increased yield. Homogenization and all the subsequent steps of the protocol must be performed at 4°C to minimize the activation of phospholipases and proteases. Avoid excessive homogenization since it can cause damage to the mitochondrial membrane and trigger release of mitochondrial components.
4. The homogenate is then centrifuged at 750 × g for 10 min.
5. The resulting supernatant is transferred to a pre-chilled centrifuge tube and stored on ice, and the pellet is resuspended in extraction buffer, homogenized and centrifuged as for steps 2–4.
6. The supernatants obtained from the two low-speed spins are pooled and then centrifuged at 10,000× g for 15 min. Crude mitochondria, which are recovered in the pellet, are resuspended in extraction buffer (approx. 20–25 μL).
7. You may check the purity and the enrichment of mitochondria from whole cells by Western blot analyses, measuring cytosolic (e.g. actin), nuclear (e.g. lamine A/C), and mitochondrial (e.g. OXPHOS subunits and porin) marker proteins.
8. The integrity of the mitochondrial fraction can be tested by quantitation of an intermembrane space protein (e.g. cytochrome c) or a matrix protein (e.g. cyclophilin D) by immunoblot. Alternatively, you could measure mitochondrial Ca2+ buffering capacity to assess the integrity of isolated mitochondria.
Most of the methods to isolate mitochondria rely on differential centrifugation, a two-step centrifugation carried out at low speed to remove intact cells, cell and tissue debris, and nuclei from whole cell extracts followed by high-speed centrifugation to concentrate mitochondria and separate them from other organelles. If you require a high degree of purity, density gradient centrifugation or affinity purification of the organelle are used to further purify mitochondria or to separate different populations of the organelle.
The paper attached below will be helpful.
Article Purification of Mitochondria by Sucrose Step Density Gradien...
Regards,
Malcolm Nobre
- Badges
- Science topic
- Similar topics
- Chemistry
- Biochemistry
- Biochemical Techniques
- Blotting Techniques
- Immunoblotting
- Dyes