Mayumi Naramura wrote: > > I'm trying to study apoptosis of > 1. fresh mouse thymocytes induced by > 2. TCR stimulation using > 3. flow cytometry, either by > 3a. Annexin V or by > 3b. TUNEL. > I know there are tremendous amount of literature related to this, but so > far I have not come across with ones that contain all the "keywords" I > listed above. Specifically, I found most of the study has been done > using "immature" T cell lines or hybridomas instead of "fresh mouse > thymocytes". > I tried it a couple of times, but I cannot see any difference between > cells stimulated with anti-CD3 and non stimulated control because a > significant portion of cells are Annexin V (+) in the control. Any > suggestions? > Mayumi Naramura, M.D. > NIH/NIAID > Lab of Immunology > Twinbrook II, Rm. 125 > 12441 Parklawn Drive > Rockville, MD 20852 > Phone: 301-402-4595 > FAX: 301-594-2522 > e-mail:mnaramura@atlas.niaid.nih.gov Mayumi, Try using anti mouse Fas. We use JO2 Mab(unlabled) as an inducer of apoptosis is fresh mouse thymocytes. It works quite well. Using 5ug/ml induces apotosis in about 30% of the cells after 1.5 hours when compared to the isotype control (hamster IgG). I am using Annexin-PI to determine apoptosis.Hope this helps. Chris Reed Senior in Genetics and Cell Biology Washington State University Pullman Washington
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