PLopez@adarc.org wrote:
> 1- In human PBMC preps, would the Annexin V (+), PI (+) fraction
> go down the drain if the prep was made using a Ficoll separation?
Inour lab, we have been able to identify annex+ apoptotic human
PBMC after ficoll. However, your question raises an importan issue
that may be of general interest. Is the cell preparatory method
selectivelyy enriching for either the apoptotic or live population? For
example adherent cultured cells undergoing apoptosis tend to detach from
the plate. If you now wash the plate, then harvest adherent cells, and
then quantify apoptosis, you will obtain an inaccurate count.
>
> 2- Is the Annexin V (+), PI(+) fraction something that might be
> unintentionally gated out if the light scatter gate is too tight ?
>
The changes which occur in celluar morphology are reflected as ligth
scatter How you draw the LS gate will definitely influence the
percentage apoptotic cells you detect by annexin. It depends on the
biological question you are asking. If your goal is to quantify total
apoptosis, then you need to try and include those events while excluding
other "noncellular" events. We have also observed that wheere the
apoptosis populatio n falls by LS can differ by mehtod of fixaiton.
Regards,
Tom
--
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Thomas W. Mc Closkey, Ph. D.
Director of Flow Cytometry, North Shore University Hospital
Assistant Professor of Pediatrics, New York University School of
Medicine
Boas Marks Biomedical Research Center, 350 Community Drive
Manhasset, Long Island, New York 11030
ph: 516-562-4844 [office], 516-562-1135/4641 [lab] fax: 516-562-2866
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