[Cytometry] Trypan Blue quenching

Elizabeth R. Simons esimons at bu.edu
Tue May 10 14:33:56 EDT 2011


Chris,
	We use Trypan blue to quench external fluorescence (bound but not  
phagocytized/endocytized organisms). You can find protocol and data  
in our paper (Bernardo et al) in Cytometry A (2010) and in my chapter  
in Current Protocols in Cytometry (ch.9.3), also 2010. I hope this  
helps.
	Liz

On May 10, 2011, at 2:27 PM, Chris Norbury wrote:

> We're trying to quantify uptake of exogenous materials (various  
> kinds) by
> flow in a heterogeneous population of cells (sorting not possible for
> biosafety reasons).  Our 37 vs 4 degree controls are not that  
> wonderful due
> to surface binding and possibly recycling, so we'd like to quench  
> surface
> fluorescence to quantify only what is internalized.  I know a lot  
> of folks
> don't go to these lengths to measure endocytosis/phagocytosis but  
> we'd like
> to.
>
> We've quenched with Trypan Blue and it seems to do a good job with  
> FITC
> labeled substrates.  BUT, it works best if present even when  
> running, rather
> than as an incubation followed by washing.  This precludes the use  
> of other
> fluorophores to identify our cell populations if Trypan Blue  
> quenches most
> other colors/dyes.  Does anyone know of the specificity of Trypan  
> Blue for
> FITC or of any other method of cell surface quenching that might be  
> more
> appropriate for us to use (we've considered acid stripping).
>
> Thanks in advance for any input.
>
> Chris
>
> -- 
> Chris Norbury, Ph.D
> Associate Professor of Microbiology and Immunology
> Penn State Milton S. Hershey College of Medicine
> Room C6764E, Dept. of Microbiology and Immunology, H107
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>
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Elizabeth R. Simons, Ph.D.
Professor of Biochemistry
Boston University School of Medicine
80 E. Concord Street
Boston, MA 02118
(617) 638-4332 phone
(617) 638-5339 FAX
esimons at bu.edu
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