--- On Sun, 2 Nov 1997 13:20:33 -0500 Mayumi Naramura <MNARAMURA@atlas.niaid.nih.gov> 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 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? When establishing a new system for the study of apoptosis,I would suggest the following: Examine the cells by fluourescence microscopy for their nuclear morphology [PI, Hoechst, AO, etc, will work]. 1 Determine what viable cells look like [your negative control; maybe just time zero cells or blocked with ICE inhibitors, zinc, etc.] and apoptotic cells look like [your positive control, maybe dexamethasone or irradiation?] 2 Perform a time course exper. to determine at what time after stimulation, apop. is detectable.The % obtained is the % apoptotic cells at the time the assay is performed. 3. With this information, now look at your experimental conditions and simultaneously examine the positve and negative controls, remembering you may need to look again a t a time course and vary the concentration of your stimulus. 4. Once you've established these control parameters, label with annexin or TUNEL and match the results with those obtained with microscopy. 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] 11/4/97 12:45:28 PM E-mail: thomasm@nshs.edu --------------------------------------------------------
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