Allo-7: a new fluorescent tandem dye for use in flow cytometry.
This report describes the development of a novel tandem dye by combining allophycocyanine (APC) and cyanine dye indotricarbocyanine (CY7) to create ALLO-7 for use in flow cytometry. The APC donor fluorophore was excited at 647 nm and, through resonance energy transfer to the CY7 acceptor, produced fluorescence at > 780 nm. To test the applicability of this tandem in single and multicolor immunofluorescence, a streptavidin conjugate of the tandem (SA-ALLO-7) was used for the detection of cell surface antigens on human peripheral blood leukocytes (PBL) by indirect immunofluorescence. Human PBL were stained with CD4/ GaM-APC, CD3-fluorescein isothiocyanate (FITC), CD14-phycoerythrin (PE), CD19-energy-coupled dye (phycoerythrin-Texas Red) (ECD), and CD8-biotin with SA-ALLO-7 and analyzed for fluorescence on a FACS Vantage using dual-laser excitation (488 and 647 nm). The results indicated that the percentage of cells positive for each of the surface antigens was comparable for single-color controls and multicolor samples. The ALLO-7 fluorescence, which was collected with a 730-shortpass dichroic mirror and a 790/50-bandpass filter, was clearly resolved from the APC fluorescence and that from FITC, PE, and ECD. The SA-ALLO-7 exhibited minimal nonspecific binding to PBL monocytes. However, the specific binding of the tandem to high-density antigens was clearly identified by positive fluorescence. This unique tandem reagent, ALLO-7, provided the capability for dual-color immunofluorescence with a 647-nm laser line (or a helium neon laser at 633 nm) and provides the potential to perform three-color analysis with a dye-head laser (Texas Red, APC, ALLO-7).
Beavis
A J
AJ
Schering Plough Research Institute, Department of Immunology, Kenilworth, New Jersey 07033, USA.
Pennline
K J
KJ
D000911:Antibodies, Monoclonal; D002232:Carbocyanines / Q000737:chemistry*; D004396:Coloring Agents / Q000737:chemistry*; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D007962:Leukocytes / Q000276:immunology; D010798:Phycocyanin / Q000737:chemistry*; D010799:Phycoerythrin / Q000737:chemistry; D013601:T-Lymphocytes / Q000276:immunology
C104268:ALLO 7; D000911:Antibodies, Monoclonal; D002232:Carbocyanines; D004396:Coloring Agents; D010798:Phycocyanin; D010799:Phycoerythrin