We tried isolating mitochondria from mouse embryonic fibroblasts using the "nitrogen bomb" method, basically according to 'Methods Enzymol. 2000;322:213-21', http://dx.doi.org/10.1016/S0076-6879(00)22022-3. After the 10 000 x g spin we were not able to see any mitochondrial pellet (which we CAN see using the traditional glass/teflon homogenizer method). We couldn't even see any cell debris in the buffer under the microscope.
We used one 150 mm plate of cells, 300 mM sucrose buffer (same as for regular mitochondria isolation). For the pressurization step, we did 30 minutes with 500 psi at 4 C, with stirring.
So, before I start with some serious troubleshooting, I was just wondering if there was anyone here who has tried this method, with or without success? What have people tried altering - pressure, time, etc?
One thing I would be careful to note with using the traditional glass/teflon homogenizer method for these purposes is that they can produce incredibly varied results. If you have the time and resources (and are determined to stay with this method for mitochondrial isolation), I would suggest doing a controlled experiment to see how many strokes are required to lyse the tissue without breaking open the cell. This will vary with user (stroke pressure/speed) and with the homogenizer used.
Before adjusting other aspects, I'd start to look at that first.
On myb Research Gate page you can find my book @Practical Mitochondriology@/ There I prese nt my original method for isolation of cell mitochondria from (so far from my experience) any type of cultured cells. It is cheap, reliable and does not requre expensice instrumentation.
Good luck/
Alexander
We tried once this:
http://www.ncbi.nlm.nih.gov/pubmed/16253339
which suggests to use 800psi for 15min.
It worked but the yield was not improved comparing to homogenizer and was lower than passing the cells through a needle >23G. Again we did it a few times and I am sure the yield can be improved by optimization for a specific cell line.
Thanks for the reply Sergei Baranov. Did you test the quality of the mitochondria? Were they well-coupled, etc?
In term of protein content it was comparable. We did not run functional tests but I suspect it is going to be similar.
Just an update for anyone who is interested:
We got this to work by decreasing the pressure to 200 psi for 30 m (10 m also seemed to work). VDAC content of the isolated mitochondria was comparable to that from our glass-teflon homogenizer method. There was no VDAC in the supernatant from the 10 000 x g spin, and there was no tubulin in the mitochondria. For some reason there was substantially less tubulin in the supernatant compared to that from the homogenizer method. Also, the cell debris pellet (1000 x g spin after the pressurization step) was consistently smaller than that from the homogenizer method.
Best of all (based on a single experiment so far), the oxygen consumption of the isolated mitochondria was good, with a state 3/state 4 ratio of 5. By comparison, the homogenized mitochondria didn't even seem to respire.
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