Re: signal detected with immunofluorescence microscopy but not with cytometry

Hi Robin,

one possibilty would be, that in the microscope the filters are  
different to your aria, so dsRed could also leak into green channel  
(like in my old microscope). So you can compare maybe single  
transfected cells and see, if the phenomenon is still there.

Second thing: Are the cells tested at almost the same time range. We  
observed for some clones, that EGFP expression peaks at 24 - 36h and  
later decreases relative rapidly, so when you look later in FACS, the  
party could be over.

Third: in rare cases we had an toxic effect of high GFP expression. So	
you will loose EGFP bright cells and detect in majority dim cells. You	
could check with 7AAD or Topro or tropo ? dyes from Molecular Probes /	
Invitrogen.

Hope this helps you a bit

Steffen

PS I know these kind of persons, who tell you, what your cytometer has	
to detect. Have you seen the cells shining bright in the microscope  
with our own eyes? Maybe they are not so bright as they hoped :-)). I  
don´t want to blame somebody, but experience is different - what a  
pitty.


Am 08.03.2006 um 22:50 schrieb Stingley, Robin L:

> Does anyone notice this with EGFP?  One of my customers swears they  
> have
> at least 20% very bright GFP in their sample (using microscopy), but
> when I run it on the Aria, less than 1% appears to be bright, and few,
> if any, are dim.  Some of the cells are also expressing dsRed, which
> shows up clearly on the Aria using the PE PMT.	The customer expected
> more trouble distinguishing the dsRed because the Aria doesn't have the
> optimal excitation, and because they felt that the dsRed wasn't as
> bright as the GFP in the first place.
>
> Any ideas would be greatly appreciated.
>
> Thanks in advance for any suggestions,
> Robin
>
> Robin Stingley, Ph.D.
> Flow Cytometry Core Facility
> Department of Microbiology and Immunology
> University of Arkansas for Medical Sciences
> Slot 511, Rm B504
> 4301 West Markham Street
> Little Rock, Arkansas 72205
> Phone:	501-686-6927
> E-mail:  StingleyRobinL@uams.edu
>
> http://www.uams.edu/flowcytometry/
>
>
>	-----Original Message-----
>	From: Julie Bertout [mailto:julie.bertout@ibl.fr]
>	Sent: Mon 3/6/2006 3:39 AM
>	To: Cytometry Mailing List
>	Cc:
>	Subject: signal detected with immunofluorescence microscopy but
> not with cytometry
>
>	Hello,
>	Can you explain me why sometimes I can detect fluorescence when
> I
>	observe cells using fluorescence microscopy but I can not detect
> it with
>	cytometry? I use different antibodies, some work for both
> technique,
>	some don't.
>	Thanks,
>	Julie Bertout
>	cytometry lab
>	Institut Pasteur de Lille
>	1 rue du professeur Calmette
>	59800 Lille
>	France
>
>
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Dr. Steffen Schmitt
FACS und Array Core Facility
NMFZ- Johannes Gutenberg Universität
Obere Zahlbacherstr. 67
55131 Mainz

Tel.: +49 6131 39 30219
Fax: +49 6131 230506

www.facslab.toxikologie.uni-mainz.de
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