>>>>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