ALLO-7: A new fluorescent tandem dye for use in flow cytometry
✍ Scribed by Andrew J. Beavis; Kenneth J. Pennline
- Publisher
- John Wiley and Sons
- Year
- 1996
- Tongue
- English
- Weight
- 543 KB
- Volume
- 24
- Category
- Article
- ISSN
- 0196-4763
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✦ Synopsis
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, CDHluorescein isothiocyanate (FITC), CD14-phycoerythrh (PE), CD1P.energy-coupled dye (phycoerythrh-Texas Red) (ECD), and CD8-bi-0th with !L4-ALLO-7 and analyzed for fluorescence on a FACS Vantage using dual-laser excitation (488 and 647 nm). The results indicated that the percent-age 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 firom 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 h e (or a helium neon laser at 633 nm) and provides the potential to perform threecolor analysis with a dye-head laser (Texas Red, APC, ALLO-7). O 19% Wlley-llsS, I~C.