chromosome sorting on aria?

James Marvin j-marvin at northwestern.edu
Mon Oct 27 15:16:49 EDT 2008


Thanks Howard.
But regarding your last comment "Also, there really aren't any other dyes 
combination other than (Hoechst dye or DAPI) and (chromomycin or 
mithramycin) that work for bivariate chromosome sorting"..

How extensively has this been looked at.  I"m sure you're aware of all the 
DNA dyes sold by invitrogen.  Unfortunately they have very little data on 
AT/GC specificity, or even DNA vs RNA specificity.  But has anybody really 
ever looked at them for chromosome sorting.  I know they have some that are 
optimally excited around the 647nm line off a krypton which I can get at 
least 500mw of.

Just wondering.  I've just heard bits a pieces of this story here and there 
and was wondering if its really been looked at.

thanks
J

At 03:29 PM 10/25/2008, Howard Shapiro wrote:
>Ben Loos wrote:
>>I trust you are all well? I would like to find out if anyone has some 
>>advice regarding chromosome
>>sorting on the FACSAria ( vilot, red and blue laser). I have seen papers
>>using Hoechst and chromomycin. I do have good experience using Hoechst
>>and the violet laser, but have no idea whether in general this could
>>work. If someone has done that on the Aria, it would be fantastic if you
>>could share experience, eg which density filter, nozzle, flow rate to be
>>used etc.
>Sorting chromosomes requires CVs in the neighborhood of 1% which are 
>impossible to achieve using a violet laser (even a relatively powerful 
>one) to excite Hoechst dyes, and usually requires at least hundreds of 
>milliwatts output from both the UV and blue (chromomycin) lasers. Somewhat 
>lower-power modelocked 355 nm lasers can be used, but you usually don't 
>find them in FACSArias. Filters, nozzles, and flow rate are irrelevant. 
>Also, there really aren't any other dyes combination other than (Hoechst 
>dye or DAPI) and (chromomycin or mithramycin) that work for bivariate 
>chromosome sorting. Sorry.
>
>-Howard

James Marvin
Manager, RHLCCC Flow Cytometry Facility
http://www.basic.northwestern.edu/sharedresources/flowcytometry/flowcyometrysite.html
312-503-0913 (office)
312-908-1294 (lab)


"What must a man possess who possesses the Possessor of all things?"
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