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Cy7PE and Cy7APC: bright new probes for immunofluorescence
M Roederer1, A B Kantor, D R Parks
1Department of Genetics, Stanford University, CA 94305-5125, USA.
Cytometry
|July 1, 1996
Summary
New indotricarbocyanine (Cy7) tandem dyes, including phycoerythrin (PE) and allophycocyanin (APC), offer enhanced fluorescence detection in flow cytometry. These novel conjugates enable sensitive, multiparametric analysis above 780 nm, even with low autofluorescence.
Area of Science:
- Biotechnology
- Immunology
- Analytical Chemistry
Background:
- Phycobiliproteins like phycoerythrin (PE) and allophycocyanin (APC) are widely used in fluorescence applications.
- Detection in the far-red spectrum (>780 nm) is challenging due to low autofluorescence.
- Development of novel fluorophores is crucial for advanced multiparametric flow cytometry.
Purpose of the Study:
- To demonstrate the utility of indotricarbocyanine (Cy7) conjugates of PE and APC as tandem dyes for flow cytometry.
- To evaluate the performance of these novel tandem dyes in fluorescence measurements, particularly in the far-red spectrum.
- To assess their suitability for immunofluorescence applications and high-order multiparametric analysis.
Main Methods:
- Conjugation of indotricarbocyanine (Cy7) to phycobiliproteins (PE and APC) to create tandem dyes.
- Excitation of tandem dyes using phycobiliprotein-specific wavelengths.
- Fluorescence measurements in the far-red spectrum (>780 nm) using flow cytometry.
- Assessment of tandem dye brightness by comparing fluorescence signal to cellular autofluorescence.
- Demonstration of a six-color immunofluorescence analysis using various fluorophores, including the novel Cy7APC tandem.
Main Results:
- Successful demonstration of Cy7-conjugated PE (Cy7PE) and APC (Cy7APC) tandem dyes for fluorescence measurements.
- Tandem dyes fluoresce above 780 nm when excited at phycobiliprotein-specific wavelengths.
- Cy7PE and Cy7APC exhibit brightness comparable to fluorescein and APC, respectively.
- These tandem dyes are easily detectable above low autofluorescence.
- The tandems are distinguishable from common fluorophores, enabling multiparametric analysis.
- A six-color analysis demonstrated the simultaneous detection of multiple fluorophores, including Cy7APC.
Conclusions:
- Indotricarbocyanine (Cy7) tandem conjugates of PE and APC are effective tools for flow cytometry.
- These novel tandem dyes provide sensitive detection in the far-red spectrum, suitable for immunofluorescence.
- Their brightness and distinct spectral properties facilitate high-order multiparametric analysis.