>
>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
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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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