I need to isolate fraction of nuclei from rodent brain and liver. Fraction must be free of ER, other membranes, mitochondria etc. (or with trace amount of other cell organelles). Can anyone help? Please don't advice kits
Dear Kateryna,
I have performed nuclei isolation for avoiding mtDNA interference in flow cytometry cell cycle analysis. Hope you will find the protocol useful for your proposes:
-Pellet the cell suspension by centrifugation at 250g, 5 min at 4 ºC (This step mainly depends on the cells you are working with).
-Resuspend the pellet in cold Nuclei Extraction Buffer (320 mM sucrose, 5 mM MgCl2, 10 mM HEPES and 1% Triton X-100; 4 ºC) and gently vortex.
-After 10 min of incubation at 4 ºC, rinse the nuclei twice by centrifugation at 2000g, 5 min at 4 ºC and resuspension in a Nuclei Washing Buffer (320 mM sucrose, 5 mM MgCl2 and 10 mM HEPES), and keep them into Nuclei Washing Buffer.
Best regards,
Dear Alex, thank you for the answer. It is very helpful. Have you tried your protocol for nuclei extraction not from cell culture but organ?
I actually performed such protocol on zebrafish cardiomyocytes directly isolated from the heart (not cultured). In this case, you just should add a cell isolating step before starting the nuclei isolation protocol, such as colagenases treatment. I am sure you will clarify this step in any primary cell culture protocol you find (suitable for your samples). Anyhow, you may keep in mind that organs are always cell populations mixtures, and so you may consider how to distinguish and/or enrich the suspension of your cells of interest.
Dear Alex, thank you for the protocol. It worked very well.
UPD. Recently, I checked the fraction with electron microscopy - only nuclei are there.
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