How to homogenize cell without disruption of nuclei?
Posted 30 April 2009 - 07:11 AM
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.
Posted 30 April 2009 - 09:19 AM
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.
Posted 30 April 2009 - 04:34 PM
I recommend getting a good protocol, there are plenty out there.