From: Jussi J. Saukkonen, MD (jsaukkonen@bupula.bu.edu)
Date: Tue Sep 22 1998 - 09:09:41 EST
>Dear Flow-ers,
>
>We started to study apoptosis 4 years ago.
>We're working with an adherent cell line and we're using flow cytometry
>detection of propidium iodide stained cells after ethanol fixation
>(apoptotic cells = sub-G1 peak). But we have lots of problems because of
>this cell line (which is adherent !), and the cell detachment is a very
>delicate step : we need to save living cells as well as apoptotic cells
>(and apoptotic bodies).
> Now we're using PBS/EDTA buffer with a very low addition of trypsin, but
>it's not the absolute solution. So we're looking for ideas in order to
>simplify this step or to substitute the method.
>Thank you for any help !
>
>Best regards,
>
>Carine MALCUS
>
>
>__________________________________________________
>
> Carine MALCUS-VOCANSON, Ph. D.
> // cellular biology // bioethics //
>
> bioMerieux S.A.
> UMR 103 CNRS - bioMerieux
> E.N.S.L.
> 46, allee d'Italie
> 69364 LYON cedex 07
> FRANCE
>
> Ph : 33 (4) 72 72 85 92
> Fax : 33 (4) 72 72 85 33
> email : Carine.Malcus-Vocanson@ens-bma.cnrs.fr
>__________________________________________________
Dear Carine,
I feel your pain, as we say here. We tried to use a PI based method on
alveolar macrophages, but had a lot of background because of detachment
trauma. We tried all kinds of plastic, coating, low adhesion surfaces to
no avail. Consider two possibilities: doing the experiment in the tubes
you will run on the flow cytometer or 2) do the experiment in multiwell
plates and do the fixation, staining and washing in the plate and then
transfer cells to the flow cytometer tubes when you are done.
Good luck,
Jussi
Jussi J. Saukkonen, M.D.
Assistant Professor of Medicine
Pulmonary Center
80 East Concord Street, R-304
Boston Univesity School of Medicine
Boston, MA 02118
Tel.: (617)-638-6120
Fax: (617)-536-8093
(617)-638-5298
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