A fluorogenic assay of endogenous phosphatase for assessment of cell adhesion
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A fluorogenic assay of endogenous phosphatase for assessment of cell adhesion. / Tolosa, E; Shaw, S.
In: J IMMUNOL METHODS, Vol. 192, No. 1-2, 10.06.1996, p. 165-72.Research output: SCORING: Contribution to journal › SCORING: Journal article › Research › peer-review
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TY - JOUR
T1 - A fluorogenic assay of endogenous phosphatase for assessment of cell adhesion
AU - Tolosa, E
AU - Shaw, S
PY - 1996/6/10
Y1 - 1996/6/10
N2 - 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.
AB - 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.
KW - Buffers
KW - Cell Adhesion
KW - Cell Line
KW - Centrifugation
KW - Chromium Radioisotopes
KW - Enzyme Activation
KW - Fluoresceins
KW - Fluorescent Dyes
KW - Humans
KW - Hydrogen-Ion Concentration
KW - Lymphocyte Count
KW - Phosphoric Monoester Hydrolases
KW - T-Lymphocytes
KW - Tumor Cells, Cultured
M3 - SCORING: Journal article
C2 - 8699013
VL - 192
SP - 165
EP - 172
JO - J IMMUNOL METHODS
JF - J IMMUNOL METHODS
SN - 0022-1759
IS - 1-2
ER -