geoscience.net logo
+ Resolve Article
+ Follow Us
Follow on FacebookFollow on Facebook
Follow on TwitterFollow on Twitter

+ Translate
+ Subscribe to Site Feed
GeoScience Most Shared ContentMost Shared Content

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


, : Detection of endogenous alkaline phosphatase activity in intact cells by flow cytometry using the fluorogenic ELF-97 phosphatase substrate. Cytometry 37(4): 314-319, Dec 1

Background: The alkaline phosphatase (AP) substrate 2-(5'-chloro-2'-phosphoryloxyphenyl)-6-chloro-4-(3H)-quinazolinone (ELF(R)-97 for enzyme-labeled fluorescence) has been found useful for the histochemical detection of endogenous AP activity and AP-tagged proteins and oligonucleotide probes. In this study, 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 chondrocytes. 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 previously described for use in detecting AP activity by flow cytometry. Results: The ELF-97 phosphatase substrate effectively detected endogenous AP activity in UMR-106 cells, with over 95% of the resulting fluorescent signal resulting from AP-specific activity (as determined by levamisole inhibition 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 high intrinsic fluorescence of the unreacted components. The ELF-97 phosphatase 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 applicability of ELF-97 to flow cytometry, supplementing its previously demonstrated histochemical applications.

US$19.90

PMID: 10547617

DOI: 10.1002/(sici)1097-0320(19991201)37:4<314::aid-cyto9>3.0.co;2-x


Other references

Yang, Y.; Li, D.; Li, C.; Liu, Y.; Jiang, K., 2017: Photoexcitation effect on the adsorption of hazardous gases on silica surface. There is very little scientific understanding of photoexcitation effect on the adsorption properties of adsorbent. The adsorption of four hazardous gases (SARIN (propan-2-ylmethylphospho-nofluoridate), methyl dichlorophosphate (MDCP), trimethyl ph...

Dhillon, T.S.; Garber, E.D.; Wyttenbach, E.G., 1961: Genetics of phytopathogenic fungi. VI. Heterocaryons involving Gibberella fujikuroi and formae of Fusarium oxysporum. Heterocaryons were obtained between mutants of G. fujikuroi and between mutants of this species on the one hand and ff. niveum, con-glutinans, pisi and lycopersici of F. oxysporutn on the other. Spores from heterocaryons had the genotype of only o...

Sadroddiny, E.; Ai, J.; Carroll, K.; Pham, T.Khoa.; Wright, P.; Pathak, A.; Helm, B., 2014: Protein profiling of the secretome of FcεRI activated RBL-2H3.1 cells. Secretory proteins of IgE receptor activated mast cells and basophils play a pivotal role in the generation of immediate and long term immune responses in allergy and type I hypersensitivity. The present study aims to generate a 2-D map and profil...

Hayashi, K.; Tanaka, H.; Miyajima, T.; Saito, K.; Kodaira, K.; Yamashita, Y., 2006: Advance in diagnosis and management guideline of AD/HD (attention deficit/hyperactivity disorder) for pediatricians in Japan. No to Hattatsu. Brain and Development 38(2): 141-143

Hausken, T.; Gilja, O.H.; Undeland, K.A.; Berstad, A., 1998: Timing of postprandial dyspeptic symptoms and transpyloric passage of gastric contents. Background: Patients with functional dyspepsia often experience early satiety and discomfort after a meal. The role of early gastric emptying in symptom generation is not known. Our aim was to relate timing of symptoms and early postprandial empty...

Silva, V.Furtado.da.; Calomeni, M.Rocha.; Nunes, R.Alkmim.Moreira.; Pimentel, C.Elias.; Martins, G.Paes.; Oliveira, Pícia.da.Cruz.Araruna.; Silva, Pícia.Bagno.; Silva, A.Pedro.Ribeiro.de.Souza.E., 2016: Brain stimulation used as biofeedback in neuronal activation of the temporal lobe area in autistic children. This study focused upon the functional capacity of mirror neurons in autistic children. 30 individuals, 10 carriers of the autistic syndrome (GCA), 10 with intellectual impairments (GDI), and 10 non-autistics (GCN) had registered eletroencephalogr...

Berg, J.W., 1977: Cancer case counts for Iowa 1973-1974. Journal of the Iowa Medical Society 67(1): 17-18

Khudik, Iag, 1978: Effectiveness of long-term application of fertilizers in a crop rotation in the mountain zone of the Carpathians. Agrokhimiia (4) 53-56

Famer, D.; Meaney, S.; Mousavi, M.; Nordberg, A.; Björkhem, I.; Crisby, M., 2007: Regulation of alpha- and beta-secretase activity by oxysterols: cerebrosterol stimulates processing of APP via the alpha-secretase pathway. The cholesterol 24-hydroxylase encoded by the gene CYP46 is expressed almost exclusively in central nervous system (CNS) neurons and catalyzes the formation of 24S-hydroxycholesterol (24S-OHC) from cholesterol. This conversion corresponds to a ma...

Haeni F.P.; Lane John W.J., 1996: Use of borehole-radar methods to detect fractures in crystalline rocks, Mirror Lake area, Grafton County, New Hampshire. Water-Resources Investigations - U S