From: Tom Mc Closkey (thomasm@nshs.edu)
Date: Thu Apr 23 1998 - 14:30:59 EST
--- On Tue, 21 Apr 1998 23:15:00 -0400 Timothy J Farley <tfarley@computer.net> wrote: >Today I was asked to help some researchers with an apoptosis assay >involving K562 cells (leukemia cell line) treated with alpha-interferon. >The procedure that they wanted to use involved treatment with 70% ethanol, >RNase, propidium iodide, and analysis of a sub G0/G1 peak. > >I know there are other methods for testing for apoptosis. Would any one >care to comment on the merits of this procedure as compared to other >methods? The method I recommend to start with is the subdiploid method. All reagents are inexpensive and it is very easy to perform the assay. For the flow analysis, I woulld suggest theat you run on a linear scale and trigger o n fluorescence so as to eliminate measuring debris. Since you are setting up a new system, a time course is a good idea to see when you can detect cell death. An additional benefit of this assay is that you can view the cells under a fluor. scope to confirm that the stimuluss is inducing nuclear changes and count apoptotic cells initally to verify the flow results. As far as comparing it to other methods, the subdiploid method compares very well. The one instance we found where it fell off was when there was massive amounts of apoptosis, it had trouble discriminating live from dead, perhaps due to free DNA sticking to cells and altering DNA content measurement. The method I consider the best as far as sensitivity, specificity, and accuracy is TUNEL. This assay is now available in kit form from several companies, but is considerably more expensive than the subdiploid method. Good Luck, Tom -------------------------------------------------------- Thomas W. Mc Closkey, Ph. D. Director, Flow Cytometry North Shore University Hospital Biomedical Research Center 350 Community Drive Manhasset, Long Island, New York 11030 ph: 516-562-4844 [office]; 516-562-1135/4641 [lab] 4/23/98 12:42:40 PM E-mail: thomasm@nshs.edu --------------------------------------------------------
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