We have a nuclear/cytoplasmic fractionation protocol that works well, but the problem is that the isolated nuclei are EXTREMELY sticky and very difficult to work with.
We want to fix these cells, stain for cell-type specific intracellular markers, and FACS sort for RNA-seq. We can do this routinely with whole cells, but isolated nuclei are totally unmanageable.
All buffers are made with Ca/Mg-free PBS, and supplementation of 5mM EDTA does not help. I have also tried treatment with 50U/ml DNase + 5mM MgCl2 to no avail.
Straining cells through a 70um nylon mesh is off the cards, as the nuclei gloops get stuck on the mesh.
Does anyone have ideas/tips and tricks to deal with this problem?
You can use a cocktail of DNase and RNAse. My experience has been to avoid EDTA and carefully control the osmotic strength of the buffer.
I found that a brief burst with a probe sonicator will break up the pellet
@Simon - hello stranger! Thanks for the tip, but do you expect that sonication would also break open the nuclei? I need them intact.
Hi there! Haha. Oh yes. The nuclei will be toast after sonication. That's a serious asterix on my suggestion. Good luck!
My experience of making individual cell suspension or keeping subcellular components in solution is quite similar to that of Michael B LoMonaco. I normally use 30 micrograms of DNase/ ml to achieve the afore mentioned goal.
The nuclear pellet should be resuspended in buffer of pH 6.9. In the slightly acidic pH the nuclei don't stick. I have used Tris-HCl buffer and with sucrose, sodium and potassium salts, traces of EDTA and EGTA etc. These nuclei were used for chromatin studies.
Hi there. I am working on unfixed nuclei separation from tissue for chromatin studies. What I found was that sucrose in the buffer is essential for the integarity of nuclei, for it helps to maintain the osmatic pressure. 5%-15% sucrose all helps. Mg++ might be also important for the integarity of nuclei. When I simply use PBS, the nuclei don't look good. The DNA was leaking and thus clump them together.
@Shuo Cao: Could you please share the protocol for the buffers/isolation procedure itself? Thank you in advance!
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