 |
 |
 |
 |
We routinely perform 5-colour surface immunophenotyping using flow
cytometry. (See Cytometry 15:371-376, 1994) using a FACS Vantage and three
excitation wavelengths (488nm, 633nm and UV 351-364 nm). The fluorochrome
combination as described in the above reference used FITC, PE and RED613
excited with the 488 line (primary beam) and APC and Cascade Blue excited
with the 633 nm and UV lines (delayed secondary beams).
Although there is a small excitation of the Texas Red component of the
RED613 tandem with the 633 laser line this should not present a problem
since the beams are spatially separated. We collected APC with a 660/20
filter but you could also try a 670/14 if you have overlap problems. Also,
this is less of a problem if you can use the 647 nm krypton line instead of
633nm.
The fluorescence signals that we acheived with Cascade blue were adequate
for resolution of high-density cell surface antigens following indirect
staining with a biotin-antibody and Neutralite Avidin-Cascade Blue. Cascade
Blue has a very unique excitation curve and depending upon your UV
wavelength being used, you may find that AMCA works just as well. Image Blue
from Leinco (ex 400, em 460) is another possibility.
One additional point, you may want to check out the abstracts for the
upcoming ISAC conference in Rimini (Beavis AJ and Pennline KP: Abstract #
AC8) as this may be of interest to you.
Regards
Andrew Beavis
Hospital For Special Surgery
Research Bldg Room 312
535 East 70th St.
New York, NY 10021
Tel. (212) 606 1925
Fax. (212) 717 1192
email. beavisa@hss.edu
|
|
|
|