A fluorogenic assay of endogenous phosphatase for assessment of cell adhesion.
Assays of cell adhesion generally require prelabeling of cells with radioactive or fluorescent probes. A new fluorogenic phosphatase assay requiring no prelabeling has been developed to quantitate cell number, which can thus serve as the basis for quantitating cell adhesion or migration. The assay uses the non-fluorescent substrate 3,6-fluorescein diphosphate (FDP) whose dephosphorylation generates fluorescein. The fluorescence generated is linear with incubation time and cell number until substrate becomes limiting; the assay easily quantitates cells over a range from 10(3) to 10(6) for a variety of cell types, including resting T cells. It is as sensitive as the 51Cr assay, but has the many advantages of a non-radioactive assay, making more convenient the removal of nonadherent cells by simple 1 x g sedimentation. Unlike most other non-radioactive assays, it requires no pre-incubation; this: (1) reduces cell manipulation; (2) eliminates problems of spontaneous release; and (3) avoids potential dye toxicity. This technique of cell quantitation has been adopted as standard in our laboratory for routine adhesion and migration assays.
Tolosa
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E
Experimental Immunology Branch, National Cancer Institute, Bethesda, MD 20892, USA.
Shaw
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S
D002021:Buffers; D002448:Cell Adhesion / Q000276:immunology*; D002460:Cell Line; D002498:Centrifugation; D002860:Chromium Radioisotopes; D004789:Enzyme Activation / Q000276:immunology; D005452:Fluoresceins / Q000032:analysis; D005456:Fluorescent Dyes; D006801:Humans; D006863:Hydrogen-Ion Concentration; D018655:Lymphocyte Count; D010744:Phosphoric Monoester Hydrolases / Q000032:analysis*; D013601:T-Lymphocytes / Q000201:enzymology* / Q000276:immunology; D014407:Tumor Cells, Cultured
D002021:Buffers; D002860:Chromium Radioisotopes; D005452:Fluoresceins; D005456:Fluorescent Dyes; C074818:3,6-fluorescein diphosphate; C085925:calcein AM; D010744:Phosphoric Monoester Hydrolases