different patterns of subG1 population

Derek Davies derek.davies at cancer.org.uk
Sun Sep 9 10:17:58 EDT 2007


Yobou,

You don't say how (if at all) these histograms have been gated. With all
subG1 analyses, you should use an area v width or an area v height plot to
exclude clumps but also to exclude small events which are likely to
represent debris etc. I then apply this gate to a scatter plot and exclude
things that are obviously not intact cells. This strategy should be applied
logically and consistently - it may also change your M1 values. As you are
seeing a G2 buildup, you should also make sure that you don't exclude
potentially reduplicating cells - and there is a hint of that in the lower
plot. You may have to reduce the voltage to bring this on scale. I might
also be tempted to look at the stained sample under a fluorescence 'scope to
see what the large percentage of dimly stained stuff was.

As has been discussed here recently, it is hard to differentiate between
apoptotic and necrotic cells on the basis of this assay and you would
probably want to use it in conjunction with for example TUNEL staining as
well as a live cell assay - annexin is one way, I quite like YO-PRO-1
staining.

Good luck,
Derek

On 7/9/07 4:03 am, "yobou" <digakuinsei at yahoo.com> wrote:

> Dear All
> the attached PDF file shows FACS analysis results of SW620 human cancer cells
> treated
> with either a death receptor ligand that induces caspase-dependent apoptosis,
> or DRUG x
> that induces caspase-independent cell death and G2 arrest. Importantly, after
> DRUG
> x-treatment no floating cells could be seen when I examined the cells under
> the
> microscope before starting FACS analysis.
>   Although the M1 marker shows close SubG1 values but the pattern or shape of
> subG1
> population is different. does such difference imply something meaningful?
>   One more question, still I did not the charachterize the type of cell death
> whether
> apoptotic-like or necrotic, is double PI/FITC-annexin staining the best way or
> should I
> use different probes?
>   sorry for displaying the results in FL2H , I greatly appreciate the comments
> of those
> who recommended me to use FL2A, but this experiment was done before I submit
> my first
> querry.
>   comments are welcome, thanks to all
> 
> ---------------------------------
> Fussy? Opinionated? Impossible to please? Perfect.  Join Yahoo!'s user panel
> and lay it
> on us.
> This attachment - 'facs querry.pdf' -  11.61 KBytes - can be viewed at
> http://www.cyto.purdue.edu/MD-parts/adf359b47940a2824d5dd2ac9226cc1c84a07105.p
> df 
> 

-- 
***************************************************************
Derek Davies, FACS Laboratory, London Research Institute,
Cancer Research UK, 44 Lincolns Inn Fields, London, UK.

Tel: (44) 20 7269 3394
FAX: (44) 20 7269 3479
mobile: 07790 604112
e_mail: derek.davies at cancer.org.uk
Web Page: http://science.cancerresearchuk.org/sci/facs/

In tenebris lux    
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