[Cytometry] Trypan Blue quenching

Chris Norbury ccn1 at psu.edu
Tue May 10 14:27:03 EDT 2011


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
500 University Drive, Hershey, PA 17033-2360
 
Lab      717 531 0624
Office   717 531 7204
Fax      717 531 6522
Skype   mhc_antigen
Email    ccn1 at psu.edu
Web      http://www.fred.psu.edu/ds/retrieve/fred/investigator/ccn1

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