We have a working tandem of allophycocyanin (APC) and indotricarbocyanine
(CY-7), APC-CY7, that has been used successfully in multicolour
immunofluorescence using a HeNe laser and a 647 nm krypton line.
I will be presenting this data at the analytical cytology session at ISAC ,
Rimini ( abstract #AC8) and a manuscript entitled " ALLO-7: A new
fluorescent tandem dye for use in flow cytometry" ( by Beavis AJ and
Pennline KP) will be published in an upcoming issue of CYTOMETRY ( vol
24(2), June 1996).
There is a significant overlap of the emission of APC with the absorbance of
CY-7 allowing for resonance energy transfer to occur between these two
fluorochromes. The near-IR emission from the CY-7 could be well resolved
from the APC fluorescence facilitating 2-colour immunofluorescence with
either the HeNe or krypton lasers and as we postulated, 3-colour
immunofluorescence analysis with a dye-head.
The APC-CY7 tandem (which we called ALLO-7) was also used for simultaneous
detection of 5 cell surface antigens by 5-colour immunofluorescence using a
FACS Vantage with 488nm and 633 nm (or 647nm) laser lines.
The ALLO-7 conjugates are quite stable since our original tandems were
prepared almost a year ago and are still providing excellent resonance
energy transfer and CY-7 fluorescence.
Although we have not had the fortune to test this new reagent on a red diode
laser, such as those used in the FACS Calibur, I see no reason why it should
not work. It would obviously provide enormous flexibilty in multicolour
immunofluorescence analysis on benchtop instruments.
I look forward to hearing your views and comments regarding this unique
tandem reagent through this service or at Rimini!
Regards
Andrew Beavis
Hospital for Special Surgery
Research Building, Room 312
535 east 70th street,
New York, NY 10021
tel. (212) 606 1925
fax (212) 717 1192
email beavisa@hss.edu