Réf. : RE: cell cycle protocol questions

zofia.maciorowski@curie.net zofia.maciorowski at curie.net
Mon Jul 4 07:15:22 EST 2005


Dear Magda,
I agree with Vincent, as the data is acquired in log, it looks like your
G1 and G2 peaks are above 10x3. You have a large peak, probably debris, at
10x1 and maybe an apoptotic peak between 10x1 and 10x3. Your  G2 appears
to be larger than your G1. This could be either doublets, as Vincent
suggests, or a tetraploid population. If you rerun the cells in log with
the PMT set so that the G1 peak is at 10x3, then switch to linear, your G1
peak will fall around channel 200 which, on a scale of 1023 channels, will
allow you to see both diploid and tetraploid populations. You can use an
area vs width plot to remove the doublets.  A good reference: chapters 3
and 8 in
Clinical Flow Cytometry Principles and Application, Ed. by Kenneth D.
Bauer,
Ricardo E Duque and T. Vincent Shankey, Pub by: Williams and Wilkins,
1993.
ISBN 0-683-00480-8
also a reference from Nathan Regimbal pulled from the email archives, with
good details on how to acquire and analyze histograms.
http://www.cyto.purdue.edu/hmarchiv/2003/att-2194/CELL_CYCLE_FUNDAMENTALS.doc

Good luck,
Zosia




"Sahi, Vincent K./Sloan-Kettering Institute" <sahiv at mskcc.org>
01/07/2005 04:20


	Pour :	Cytometry Mailing List <cytometry at flowcyt.cyto.purdue.edu>
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	Objet : RE: cell cycle protocol questions


Magda,



I don't think your problem is your protocol; it's your data acquisition.
Did you acquire your data in Log? If so, then it looks to me that your g1
and g2 peaks are the second set above 10x3. Did you gate out the doublets?





Vincent Sahi

Memorial Sloan-Kettering



-----Original Message-----
From: Magda Lezcano [mailto:magda.lezcano at ebc.uu.se]
Sent: Wed 6/29/2005 3:38 PM
To: cyto-inbox
Cc:
Subject: cell cycle protocol questions


Dear followers, Im trying to do cell cycle analysis and I have got funny
graphs, Im getting two picks at G2 and one of them is sometimes quite
higher even than the G1, besides they are also the s-phase seems to be
quite wide to me, so if anyone have any suggestion what could be my
problem please let me know or if someone have any good protocol, please
also forward it to me... thanks MAgda


--
Magda Lezcano
Department of Animal Development and Genetics
EBC, Uppsala University
Norbyvägen 18A, 75236
Uppsala, Sweden
tel. +46-184712679
Fax. +46-184712683
mobile: 0735351834


"When joy is at its highest
Sad thoughts run rife
Youth and strength, how short they last
How hopelessly we age!"

Emperor Han Wudi, Western Han Dynasty

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