I am looking for cheaper/homegrown options for reliable yields of intact minipig mitochondria.
I am interested in the cell phenotype and the mito stress tests, preferably using Seahorse.
Also, I would highly appreciate it if there are any suggestions/comments to someone working for the first time using these protocols.
Chapter Isolation of mitochondria from cells and tissues
Article Mitochondria isolated in nearly isotonic KCl buffer: Focus o...
Chapter Isolation of mitochondria from cells and tissues
Those may be helpful. Good luck.
Dear Lalitha,
Please You look at the protocol:
Article A transgenic minipig model of Huntington's disease shows ear...
Minipig material and sample collection
For isolation of the mitochondrial fraction, 5% homogenate (W/V) was prepared from the frozen tissue using Potter-Elvehjem homogenizer in KTEA medium (150 mM KCl, 50 mM Tris-HCl, 2 mM EDTA, pH 7.4, 0.2 μg/ml aprotininin) at 40°C. Mitochondria were sedimented by centrifugation of 600 g postnuclear supernatant at 10,000 g for 10 min at 4°C. The pellets were washed with KTEA medium and centrifuged again in the same conditions. Finally, the pellets were resuspended in KTEA medium to a protein concentration of ∼20 mg/ml. Measurement of mitochondrial enzymes was performed immediately after isolation. For western blotting and measurements of PDH activity, frozen aliquots were used.
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