isolating adipocytes for counting purpose - (Jul/27/2006 )

Hello everyone, newbe here with some questions. I did a quick search but did not find any posts that answered my questions.

I am trying to isolate human adipocytes from the large globules they are in, and then stain them with trypan blue to count the viable ones in a hemacytometer. This will be my first time doing this. I have read different protase treatments, and some seem very elaborate. I would like to do something very simple. Treatment with Trypsin or collagenase seem to be the most popular. The trypsin method seems to be the easier of the two and I will try that one first. (unless someone here points me in a better direction) Here are my questions.

1. Where would I be able to get a step by step instructional protocol/procedure that I can follow for either the trypsin or collagenase treatments? or for both?
2. Would this protocol/procedure contain the part numbers or where to buy all the different materials necessary for these treatments. For example, I know I can get collagenase from Sigma Aldrich, but they carry something like 50 different kinds, how do I know which one to buy?
3. Is there a better way to isolate the human adipocytes than with the two treatmens mentioned above?

Thanks in advance for the help.

T

Would this help? http://www.serva.de/products/sheets/Prot_a...s_pre_human.pdf

This is great help. Thank you very much. Anything silimar to this for the Trypsin treatment? I think the trypsin treatment requires less solutions, buffers, etc. and would be easier to perform. However, it also seems that the collagenase treatment would yeild better results. Is this about right or am I way off base? I still would like to perform the easier protocol first and work my way up to the more difficult one. Does that make sense too? Thanks again, any help is greatly appreciated!

To be honest with you, I have not done any isolation of these cells. Clloagenase treatment would be milder to cells, I think.

Well, tried the trypsin treatment and it didn't seem to help much. I will have to go back to double check to make sure I didn't do something wrong... Other than that, I am going to attempt to do the collagenase treatment. We'll see how it goes. Thanks for the help.