[Cytometry] Flow karyotyping

Derek Davies derek.davies at cancer.org.uk
Fri Jul 17 03:55:42 EDT 2009


Hi Jeff,

Being able to identify and sort a derivative chromosome will depend on it
having a different size (DNA content) or bp ratio such that it would move
from its normal position on a flow karyotype. Small changes, reciprocal
translocations and so on are often not seen. Traditionally (and we haven't
sorted a chromosome in anger for quite a while) we used H33258 and
chromomycin A3. As you say chromomycin is best excited using a 457nm line (a
rather attractive blue) and I recall using around 300mW of that and UV to
get best resolution, the more you put in with small stuff the better the
signal and CV. Havent tried with a violet laser but I suspect the power
output wouldn't give you the required resolution. I think there have been
papers using 488nm excited dyes but again I don't think they work that well
and the Hoechst/chromomycin combination is still the gold standard.

Best wishes,
Derek



On 16/7/09 08:55, "Jeff Barry" <JBarry at picr.man.ac.uk> wrote:

> Hello,
> 
>  
> 
> We have a researcher who would like to resolve and sort human
> isochromosome 17q.  From the literature I found that a bivariate plot of
> Hoechst against Chromomycin A3 seems to resolve chromosome 17 nicely but
> does anyone know if the i(17q) will form a distinct separate population?
> 
>  
> 
> I'm also concerned that our present sorters' configurations may not be
> ideally suited to this, especially regarding the choice of laser lines
> and power. We have an Influx with 100mw UV and 50wm 407nm laser. I
> understand that the laser line of choice for Chromomycin is 457nm but
> has anyone tried with a 407nm line?
> 
>  
> 
> We also have a Vantage that has a UV laser (<100mw) and an Argon ion
> laser that could in theory be tuned to 457nm (100wm).
> 
>  
> 
> Does anyone have an opinion whether these sorters could be used?
> 
>  
> 
> If anyone has done this before and is willing to share their experience
> I would be very grateful.
> 
>  
> 
> Jeff Barry
> 
> Cancer Research UK
> 
> Paterson Institute
> 
> Manchester University
> 
> England 
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-- 
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Derek Davies, FACS Laboratory, London Research Institute,
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