Abstract | BACKGROUND: The fluorogenic alkaline phosphatase (AP) substrate 2-(5'-chloro-2'-phosphoryloxyphenyl)-6-chloro-4-(3H)-quinazolinone (ELF( R)-97 phosphate, for Enzyme-Labeled Fluorescence) has been used primarily in microscope-based imaging applications to detect endogenous AP activity, antigens and various ligands in cells and tissues, and nucleic acid hybridization. In a previous study, we demonstrated the applicability of ELF-97 phosphate for detecting endogenous AP activity by flow cytometry. In this study, we show that the spectral characteristics and high signal-to-noise ratio provided by the ELF-97 phosphate make it a useful label for immunodetection via flow cytometry. It can be combined with a variety of other fluorochromes for multiparametric flow cytometry analysis of both endogenous AP activity and intracellular and extracellular immunolabeling with AP-conjugated antibodies. METHODS: RESULTS: Application of the ELF-97 phosphate to detect AP was found to be compatible with immunodetection and DNA staining techniques. It was also spectrally compatible with a variety of other fluorochromes. Endogenous AP activity could be detected simultaneously with both intracellular antigens labeled using Oregon Green 488 dye, PE, Cy5 dye and Alexa Fluor 568 dye-conjugated antibodies, and DNA content analysis with PI or 7-AAD. This multiparametric assay accurately delineated the distribution of AP in cycling cells and was able to identify cell subsets with varying endogenous AP levels. The ELF-97 alcohol reaction product was found to be an effective label for intracellular antigen immunolabeling with AP-conjugated reagents, and could also be combined with PI and 7-AAD. ELF-97 phosphate was also found to be a useful label for extracellular antigen immunolabeling with AP conjugates, and was compatible with Oregon Green 488 dye, PE, and PE-Cy5 dye-labeled antibodies for four-color surface labeling with minimal spectral overlap and color compensation. CONCLUSIONS:
ELF-97 phosphate was shown to be a useful label for both endogenous and antibody-conjugated AP activity as detected by flow cytometry. Its spectral characteristics allow it to be combined with a variety of fluorochromes for multiparametric analysis. Cytometry 43:117-125, 2001. Published 2001 Wiley-Liss, Inc.
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Authors | W Telford, W Cox, V Singer |
Journal | Cytometry
(Cytometry)
Vol. 43
Issue 2
Pg. 117-25
(Feb 01 2001)
ISSN: 0196-4763 [Print] United States |
PMID | 11169576
(Publication Type: Journal Article)
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Chemical References |
- Antibodies
- Antigens
- DNA, Neoplasm
- Fluorescent Dyes
- Organophosphorus Compounds
- Quinazolines
- Quinazolinones
- 2-(5'-chloro-2'-phosphoryloxyphenyl)-6-chloro-4-(3H)-quinazolinone
- DNA
- Alkaline Phosphatase
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Topics |
- Alkaline Phosphatase
(analysis, metabolism)
- Animals
- Antibodies
(metabolism)
- Antigens
(analysis, immunology)
- Cells, Cultured
- DNA
(analysis)
- DNA, Neoplasm
(analysis)
- Extracellular Space
(enzymology)
- Flow Cytometry
(methods)
- Fluorescent Dyes
(metabolism)
- Intracellular Fluid
(enzymology)
- Male
- Mice
- Mice, Inbred BALB C
- Organophosphorus Compounds
(metabolism)
- Quinazolines
(metabolism)
- Quinazolinones
- Rats
- Tumor Cells, Cultured
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