Dear Dan, <<Cell Cycle gating>> I don't know how you stained the cells (is it still cells or just nuclei??) It looks to me that the events you are referring to in your figure are not apoptotic events but just fluorescent debris (maybe some fibrous material, hence their large width signal, I have marked them wit a red arrow on the figure). A real sub-G1 apoptotic population can only be reliable quantified if your staining protocol is especially developed for that: it should start not far down from G1 events towards lower fluorescence, i.e. the ones I have marked with a green arrow on the figure. I hope this helps. Best Regards, Dirk Prof. Dirk Van Bockstaele, PhD Antwerp University Hospital Laboratory of Hematology Head of Flow Cytometry & Molecular Diagnostics Wilrijkstraat 10 B-2650 Edegem Belgium. phone: 32 3 821 3900, fax 32 3 825 1148 e-mail: dirk.van.bockstaele@uza.be > ---------- > Van: Rosson, Dan[SMTP:RossonD@MLHS.ORG] > Verzonden: woensdag 4 september 2002 15:02 > Aan: Cytometry Mailing List > Onderwerp: Gating for apoptosis studies > > <<Bestand: Cell Cycle gating.ppt>> > Dear Fellow Flowers: > > I'd like to ask my more learned flowlosopher colleagues a question on > gating for the purpose of quantitating sub G1 content for apoptosis > studies. I'm hoping Powerpoint illustrations can be attached to these mass > mailings. We all know that we should gate out doublets and triplets in > order to restrict a cell cycle analysis to single cells and one of the > ways of doing this is to plot Area vs Width in order to draw a region. The > Powerpoint attachment is a dot plot of such an experiment. Notice the long > trail of sub G1 events that are big, fat and wide. Should these events be > gated out as in the first histogram or left in as in the second histogram? > It seems to me that if they are two or more sub G1 events are stuck > together they're still apoptotic and should be left in the gate otherwise > it's cheating the apoptotic population of its due representation. However, > I've never seen a discussion of this. So, if some of you have any insight > into this, I'd like to hear it. > > Sincerely, > > Dan Rosson > > Dan Rosson Ph.D. > Lankenau Institute of Medical Research > 100 Lancaster Ave. > Wynnewood, PA 19096 > www.limr.org. > >
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