How to homogenize cell without disruption of nuclei? - (Apr/30/2009 )
I would a few questions concerning cell homogenization.
If I want to disrupt cells, but I do not want to disrupt nuclei, which type of homogenisator-mechanical homogenization can I use?
I know two types of homogenisator for mild homogenization – Dounce homogenisator and Potter-Elvehjem homogenisator. Dounce h. it is glass tube with glass pestle and Potter-Elvehjem h. is glass tube with teflon pestle. We have in lab P-E h., so I used it for homogenization of cells scraped in PBS, after 30 strokes with the pestle I could see in cell homogenizate under the microscope many dark circles that, I suppose, were nuclei but may be not, and it were non disrupted cells? I was not sure, whether homogenization was very successful or not.
Thus my next questions are:
Are those both homogenisators usefull for cell homogenization without disruption of nuclei? What are your experiences? How can I recognize in normal microscope that, what I see, are nuclei, i.e. that homogenization was successful?
Thanks in advance.
How about a chemical lysis?
Mechanical means may be hard to gauge, so I would recommend a mild chemical breakage of cell membrane. Searching the internet should give you a lot of ways to isolate nuclei.
Most cellular fractionation protocols use a combination of hypo-osmotic solution to swell the cells and then a mechanical disruption. You will need a pestle that has a short round bulb on the end - such as this dounce one, rather than a cylindrical one like this one. The cylindrical ones provide too much area against the cell so they rupture fully. You also have to be careful on the clearance between the pestle and mortar; too tight and it will disrupt the whole cell, too loose and it won't work.
I recommend getting a good protocol, there are plenty out there.