PerCP chemistry

tony_ward@bdis.com tony_ward at bdis.com
Mon Jun 26 15:25:17 EST 1995


Frederic I. Preffer writes:

>>>>Can someone please outline  the physical attributes of PerCP, with respect
>>>>to that dyes requirements for excitation time, laser power requirements,
>>>>etc. How do these compare/contrast with the requirements of something like
>>>>FITC, PE, or APC?

>>>>For example, I see that PerCP  works well when exited within the cuvette
>>>>of a FACScan, but not in a stream-in-air sorter,  ostensibly due to the
>>>>longer time and lower laser-power requirements of the dye.  Can someone give
>>>>me the real FACS? thanks.

PerCP is approximately 35Kd in size and has the following properties:

                    FITC        PE          PerCP       APC
Excitation (max):   495nm       565nm*      470nm       650nm
Emission (max):     525nm       575nm       677nm       660nm
Optimal excitation: 80mW        80mW        <15mW       460 mW**
Excitation time:    ***see below
Quantum Yield:      ?.65        ?0.94       No data       ?.75

*PE has a soret band maxima at 470nm, which enables the 488 excitation.
**APC S/N at 460mW only twice the S/N obtained at 47mW when 633 nm excitation 
was used (Loken et al, Cytometry 8:96-100 (1987).
***I don't have any specifics - but I think there may be some info in a chapter 
written by Alan Waggoner, "Fluorescent Probes for Cytometry", Flow Cytometry and 
Sorting, 2nd Ed. Melamen, Lindmo, and Mendelsohn Eds., Wiley Liss (1990) pp. 
209-225.

Relative Spectral Overlap*:

                    FITC        PE          PerCP       APC
FITC                1.000       0.200       0.003       0.000
PE                  0.015       1.000       0.080       0.000
PerCP               0.000       0.000       1.000      0.05
APC                 0.000       0.000      0.05        1.000           

*Filter choices will of course change the relative amounts of compensation 
required.

FYI: the molecule is extracted from a dinoflagellate.  By contrast, PE and APC 
are extracted from the phycobilisomes of either blue-green bacteria or various 
rhodophytes.  

Having said that PerCP is optimal on <15mW, if a 50mW laser on a stream in air 
is used with appropriate filters, it is possible to use PerCP for highly 
expressed antigen detection.  NOTE: I wouldn't try and use HLA-DR PerCP on a 
vantage!

I hope that this helps

Tony_ward at bdis.com




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