I have stored the tissues in -80 degree. I need to check the activity of mitochondrial enzymes and Calcium accumulation. I want to clear, how long we can store the tissues, whether mitochondria is stable in stored tissues.
I would expect that the enzymes will be fully active, but the functions depending on the membrane integrity are lost. This will be caused by the freezing itself rather than the duration of storage.
In vitro measurements of individual respiratory chain complexes I-IV can be performed using frozen samples according to the attached publication which is, by the way, an excellent resource for assaying these complexes.
Cheers, Christian
Article Assessment of mitochondrial respiratory chain enzymatic acti...
It is possible to isolate and perform mitochondrial complex activities on snap-frozen tissues with slight loss of complex activities. But it is always better to perform on fresh tissues. It is also recommended to do a quick analysis of complex activities using snap-frozen and fresh tissue of interest for comparison. Complex I activities should be done on the same day of mitochondria isolation. The reference cited by Christian is an excellent protocol article for mitochondrial complex enzymes.
However, mitochondrial functional assays (respirometry for example) should be done on freshly isolated mitochondria.
Yes, it is possible to isolate mitochondria from frozen tissue. Tissues can be stored for more than 7 years in -80 degree, but isolated mitochondria can be stored for 2 years only. Yes, mitochondria is stable in stored (-80 degree) tissues.
Important points to be considered to check mitochondrial enzyme activity are
(1) Post mortem interval (PMI) of tissue (more the PMI the more chances to see a reduction in the activity)
(2) how many times the tissue has undergone freeze-thaw. Generally, we check the activity of mitochondria at different freeze-thaws, we find more activity at 3rd freeze-thaw. If it is more than that we see reduction in mitochondrial enzyme activity.
For more information on mitochondrial enzyme assays refer to the attachment and also check other papers from the same team.
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