[Cytometry] Doube negative off red laser path

Rachael Walker rvw24 at cam.ac.uk
Fri Aug 13 07:54:30 EDT 2010


Hi Julie,

I think I see a similar thing on my MoFlo, which I have posted as a 
question on the Purdue list before.  I see what has been described to me 
as a 'ghost population'.  I get a second population of 'negative' on the 
axis when I use my 561nm laser.  I occasionally see it with my 635nm 
laser.  As with your population when I open the illumination chamber the 
population disappears.  I would be keen to find out exactly what the 
extra population is so that I can explain it to my users. 

Best Wishes

Rachael



Julie GOLDEN Nelson wrote:
> Hi y'all,
>
>  I've been using a Melles Griot 633 laser for 9 years now.  For it's entire life on my MoFlo Legacy and now on my MoFlo XDP, I have gotten 2 negative peaks when I run cells - I get just the one negative with beads.  At the beginning of my sort career, a flow person for whom I have great respect explained this by saying that the red laser does not efficiently excite autofluorescence and this explains the double peak - some cells are excited "better" than others.  To prove it, he shined a flashlight into the sort chamber and miraculously the double peak turned in to one.  This has always been a satisfactory explanation for me but lately my users have not been buying it.  Mainly because it does not happen upon excitation with red lasers on all of my other cytometers.  So my question is twofold - does anyone else out there witness this phenomena and is this a satisfactory explanation?  (Cells stained with APC and Alexa Fluor 633 are brilliant - far out in the 3rd or 4th decade d!
>  epending on the antibody and easily separable from the double negative.)  If you want a picture, I can send you one.
>
> Thank you all for your patience and time,
>
> Julie
>
> Julie Nelson
> Flow Cytometry Core Facility
> Center for Tropical and Emerging Global Disease
> University of Georgia
> jnelson at uga.edu
> 706-542-9474
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-- 


Dr Rachael Walker
Flow Cytometry Core Facility Manager
Wellcome Trust Centre for Stem Cell Research
University of Cambridge
Tennis Court Road
Cambridge
CB2 1QR

01223 760227





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