Re: macrophage apoptosis and flow

From: Jane S. Miller (miller@medicine.tamu.edu)
Date: Thu Apr 20 2000 - 08:14:40 EST


I'm wondering why you are using FL3 instead of FL2 for propidium iodine?  Would that
effect the brightness?

>>> Maciej Simm <simmmmer@yahoo.com> 04/18/00 05:14PM >>>

Dear all,

One of the people I'm working with right now is studying apoptosis in
periteneal macrophages using a kit with annexin V monoclonal FITC ab
and propidium iodine.

The cells are autofluorescent. The "unstained" tube has signal in up
to 2nd log.

The stained tubes are VERY HIGHLY BRIGH on FL3 (PI) and I tried to
make things "fit" on the dotplot by lowering fl3 voltage by 200 or
so, but I'm still not getting everything (and also some of the dimmer
cells are squooshed on the axis). The FITC signal is of normal
brighness.

Needless to say we're not getting the same results as the kit
suggests we should given the incubation/conditions. He's gonna talk
to the company. I am posting this message with hopes that some of you
may have seen this method or one similar to it and are willing to
comment.

Any suggestions are welcome

thanks a bunch,

Maciej



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