Re: cell cycle analysis : move 2n peak?
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From: <vincent.shankey@[EMAIL PROTECTED]
>
Date: Thu Dec 14 2006 - 12:17:42 EST
Slight changes in peak channel for 2N populations can be due to
instrumentation, staining methodology, and/or actual variations in 2N
DNA
content for different cell populations. As was pointed out in the DNA
Cytometry Guidelines (Cytometry 1993, 14;472-477),, slight changes in
G1
peak position can be caused by relatively small changes in fixation
conditions, cell concentration and/or dye concentration. It might be
useful to fix and stain replicate samples and determine the
variability of
G1 peak position due to methodology (using beads to standardize
instrument
performance). It was argued in the DNA Cytometry Guidelines that
addition
of other types of cells (chicken, trout, etc) or mixing the sample
with
other types of cells generally causes uncertainty regarding diploid
G1
peak position for the reasons mentioned above. If the variation seen
is
likely due to sample processing (and this is not a clinical sample),
then
you can either live with the variability in the measurement, or adjust
the
high voltage (to keep the G1 channel the same) to keep the data
"visually
acceptable" to people who do not understand experimental fluctuations
(including some reviewers).
T. Vincent Shankey, Ph.D.
Advanced Technology Center
Beckman Coulter, Inc.
vincent.shankey@[EMAIL PROTECTED]
(305)-380-2430
Yoav Altman <yoav@[EMAIL PROTECTED]
>
12/12/2006 02:25 PM
To
Cytometry Mailing List <cytometry@[EMAIL PROTECTED]
>
cc
Subject
Re: cell cycle analysis : move 2n peak?
Julie,
If you want to create overlay figures for presentation or
publication, you'll probably want the 2n peaks to all line up in the
same channel.
To rule out the possibility that movement of the 2N peak is due to a
difference in DNA content between two samples you can take a small
aliquot from each sample, mix them together in the same tube, let
them equilibrate for a minute or two and then run on the cytometer.
Basically, you are creating an in-tube overlay. If the two samples
have 2N peaks with differing DNA content (and your CVs are low
enough) you'll see a pair of 2N peaks in the mixed tube.
Once you confirm that your samples have 2N peaks with the same DNA
content you should feel comfortable adjusting the voltage on a sample
by sample basis to place the 2N peak at channel 200.
Another option is to spike each sample with a control like Trout
Erythrocyte Nuclei (TEN) and adjust PMT voltage to place the TEN peak
in the same channel for each sample.
Regards,
Yoav
At 4:38 PM +0100 12/12/06, Julie Bertout wrote:
>Hello,
>I have a question about cell cycle analysis using IP :
>a researcher is interested in studying the effect of drugs on cell cycle.
>He is doing IP staining on cells after different treatments.
>I adjusted the peak 2n at 200 with the control cells but with some
>conditions the peak 2n ****fts a little bit (higher or lower
>depending on the condition) so my question is :
>do I have to adjust the peak at 200 for each conditions (so we can
>do an overlay after) or keep the same cyto settings and adjust the
>marker?
>
>Thank you for your answers
>
>Julie Bertout
>cytometry lab
>Institut Pasteur de Lille
>1 rue du professeur Calmette
>59800 Lille
>France
--
Yoav Altman
Manager, High Throughput Cell Analysis Shared Resource
Burnham Institute for Medical Research
10901 North Torrey Pines Road, La Jolla, CA 92037
(858) 646-3100 x3569
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Received on Fri Dec 15 13:18:00 2006
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