--- On Wed, 17 Dec 1997 21:33:58 -0500 (EST) Bob Donahoe <rdonaho@emory.edu> wrote: > > No surprises here, I'm lost and confused. What is the best way to >analyze apoptosis in circulating leukocytes by cytofluorometry, especially >in regard to AIDS viral infection? Thanks in advance and happy holidays. You have a number of choices. The method where you look for cells with subdiploid DNA content is the easiest cell prep but can be tricky to analyze correctly. You need to be careful with instrument and gate settings in order to not include debris and thus overstimate the % apo. TUNEL is a highly recommended method and has become easier since it is now available in kit form. Annexin seems to be gaining in popularity. There is no control molecule available, so keep this in mind. Apo 2.7 is easy to use, just a cytoplasmic Ab label. Other assays that require live cells, such as the Hoechst/7-AAD method and methods looking at mitochondrial function, are a bit more complex. I would suggest starting with the subdiploid method. Its a good idea to confirm any assay in your system, for this I would recommend fluorescence microscopy and actually count apop cells after staining the nucleus. You will see some of the classic characteristic s of this process. Verify the #s under th e scope match those generated by the cytometer. A cell culture period is required to see decent numbers, we use 3 day culture as determined by a time course done a few years back. Patients at different stages of disease will show different levels of cell death and bear in mind that treatment may also be a variable to consider. Also in question is whether you want to look at spontaneous or induced cell dath and what stimulus to use. 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] 12/19/97 12:40:02 PM E-mail: thomasm@nshs.edu --------------------------------------------------------
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