Hey guys,
i would like to know, if there is any existing protocol, how to isolate mitochondira from treated cells and use them further in Seahorse XFP Mito Stress test for 8well plates? Second question is how to check, if i really isolated only mitochondria, as i cant see them? My suggestion would be mito tracker, but maybe you have an easier way?
Hi,
Below is how I isolate mitochondria from MEFs for enzymatic assays. I hope you find it helpful.
- Homogenize the cells in HB buffer (250mM Sucrose; 2mM Hepes; 0.1mM EGTA) using a Teflon pestle (1,600 r.p.m, about 40 strokes)
- Transfer the homogenate to a 15 ml tube and centrifuge at 600g for 10 min at 4 °C.
- Collect the mitochondria-rich supernatant, transfer it to a new centrifuge tube and centrifuge at 10,000g for 10 min at 4 °C to pellet mitochondria.
To check the purify of mitochondrial extracts, you can do Western blots using antibodies against common mitochondrial and cytoplasmic protein markers, for example, porin and tubulin.
Good luck!
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