Detection of endogenous alkaline phosphatase activity in intact cells by flow cytometry using the fluorogenic ELF-97 phosphatase substrate

Background: The alkaline phosphatase (AP) substrate 2-(5'-chloro-2'-phospho ryloxyphenyl)-6-chloro-4-(3H)-azolinone (ELF(R)-97 for enzyme-labeled fluor escence) was been found useful for the histochemical detection of endogenou s AP activity and AP-tagged proteins and oligonucleotide probes. In this st udy, we evaluated its effectiveness at detecting endogenous AP activity by flow cytometry. Methods: The ELF-97 phosphatase substrate was used to detect endogenous AP activity in UMR-106 rat osteosarcoma cells and primary cultures of chick ch ondrocytes. Cells were labeled with the ELF-97 reagent and analyzed by flow cytometry using an argon ultraviolet (UV) laser. For comparison purposes, cells were also assayed for AP using a Fast Red Violet LB azo dye assay pre viously described for use in detecting AP activity by flow cytometry. Results: The ELF-97 phosphatase substrate effectively detected endogenous A P activity in UMR-106 cells, with over 95% of the resulting fluorescent sig nal resulting from AP-specific activity (as determined by levamisole inhibi tion of AP activity). In contrast, less than 70% of the fluorescent signal from the Fast Red Violet LB (FRV) assay was AP-dependent, reflecting the hi gh intrinsic fluorescence of the unreacted components. The ELF-97 phosphata se assay was also able to detect very low AP activity in chick chondrocytes that was undetectable by the azo dye method. Conclusions: The ELF-97 phosphatase assay was able to detect endogenous AP activity in fixed mammalian and avian cells by flow cytometry with superior sensitivity to previously described assays. This work also shows the appli cability of ELF-97 to flow cytometry, supplementing its previously demonstr ated histochemical applications. Cytometry 37:314-319, 1999. (C) 1999 Wiley -Liss, Inc.