Re: cell cycle analysis : move 2n peak?

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@coulter.com
(305)-380-2430




Yoav Altman <yoav@burnham.org> 
12/12/2006 02:25 PM

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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 shifts 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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