Detection of endogenous and antibody-conjugated alkaline phosphatase with ELF-97 phosphate in multicolor flow cytometry applications.
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
ELF-97 phosphate detection of endogenous AP activity in UMR-106 rat osteosarcoma cells was combined with intracellular antigen detection using Oregon Green 488 dye-conjugated secondary antibodies and DNA content analysis using propidium iodide (PI) or 7-aminoactinomycin D (7-AAD). ELF-97 phosphate detection of endogenous AP was also tested for spectral compatibility with a variety of other commonly used fluorochromes. ELF-97 phosphate was then used to directly label intracellular antigens via AP-conjugated antibodies, again combined with the analysis of DNA content using PI and 7-AAD. ELF-97 phosphate was also used to directly detect extracellular antigens. It was combined with Oregon Green 488 dye, phycoerythrin (PE), and PE-Cy5 dye-labeled antibodies for simultaneous four-color analysis. All samples were analyzed on a dual-beam flow cytometer, with UV excitation of the ELF-97 alcohol reaction product.


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.
Telford
W
W
Experimental Transplantation and Immunology Section, Medicine Branch, Division of Clinical Sciences, NCI-NIH, Bethesda, Maryland, USA. telfordw@box-t.nih.gov
Cox
W
W
Singer
V
V
D000469:Alkaline Phosphatase / Q000032:analysis* / Q000378:metabolism; D000818:Animals; D000906:Antibodies / Q000378:metabolism*; D000941:Antigens / Q000032:analysis / Q000276:immunology; D002478:Cells, Cultured; D004247:DNA / Q000032:analysis; D004273:DNA, Neoplasm / Q000032:analysis; D005110:Extracellular Space / Q000201:enzymology; D005434:Flow Cytometry / Q000379:methods*; D005456:Fluorescent Dyes / Q000378:metabolism*; D007424:Intracellular Fluid / Q000201:enzymology; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D009943:Organophosphorus Compounds / Q000378:metabolism*; D011799:Quinazolines / Q000378:metabolism*; D052999:Quinazolinones; D051381:Rats; D014407:Tumor Cells, Cultured
D000906:Antibodies; D000941:Antigens; D004273:DNA, Neoplasm; D005456:Fluorescent Dyes; D009943:Organophosphorus Compounds; D011799:Quinazolines; D052999:Quinazolinones; C079177:2-(5'-chloro-2'-phosphoryloxyphenyl)-6-chloro-4-(3H)-quinazolinone; D004247:DNA; D000469:Alkaline Phosphatase