Determination of the adhesive capability of canine polymorphonuclear neutrophil granulocytes using a fluorometric microtiter plate cellular adhesion assay

A rapid fluorometric cellular adhesion assay in microtiter plates was evaluated for canine polymorphonuclear neutrophil granulocytes (PMN) using the fluorescent indicator calcein acetoxymethyl ester (Calcein AM). Optimum adhesive responsiveness occurred in bovine serum albumin (BSA)-coated plates af...

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Bibliographic Details
Published in:Veterinary immunology and immunopathology 1999-05, Vol.68 (2), p.283-291
Main Authors: Petersen, Thomas Kongstad, Væver Bysted, Britta, Jensen, Asger Lundorff
Format: Article
Language:English
Subjects:
Adhesion-assay
Adhesiveness
ANIMAL DE LABORATOIRE
ANIMALES DE LABORATORIO
Animals
Calcein AM
Canine
Cell Adhesion
CHIEN
DOGS
Dogs - blood
Female
Fluoresceins - metabolism
FLUORESCENCE
FLUORESCENCIA
Fluorescent
Fluorescent Dyes - metabolism
Fluorometry - veterinary
GRANULOCITOS
GRANULOCYTE
GRANULOCYTES
INFLAMACION
INFLAMMATION
LABORATORY ANIMALS
Leukocyte Count - veterinary
Male
Microtiter plates
Neutrophil Activation - drug effects
Neutrophil Activation - physiology
Neutrophils
Neutrophils - metabolism
PERRO
Tetradecanoylphorbol Acetate - pharmacology
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Description
Summary:A rapid fluorometric cellular adhesion assay in microtiter plates was evaluated for canine polymorphonuclear neutrophil granulocytes (PMN) using the fluorescent indicator calcein acetoxymethyl ester (Calcein AM). Optimum adhesive responsiveness occurred in bovine serum albumin (BSA)-coated plates after 30 min incubation at 37°C with 5 × 10 5 PMN in 100 μl per well using phorbol myristate acetate (PMA, 100 ng/ml) as stimulant. The adhesive responsiveness for 15 laboratory beagle dogs was determined and the mean percentage of adherence for unstimulated (2.9%) and stimulated (74.3%) PMN and corresponding 95% confidence intervals was determined. The mean intra-assay coefficient of variation (CV intra) for unstimulated and stimulated adhesion in the assay was: CV intra = 12.9% and CV intra = 4.1%, respectively. The mean inter-assay coefficient of variation (CV inter) for unstimulated and stimulated adhesion, respectively, in the assay was: CV inter = 22.9% and CV inter = 5.6%. The assay was highly reproducible and labelling with Calcein AM neither reduced PMN viability nor activated the PMN.
ISSN:0165-2427
1873-2534
DOI:10.1016/S0165-2427(99)00016-1