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 evalu ated for canine polymorphonuclear neutrophil granulocytes (PMN) using the f luorescent indicator calcein acetoxymethyl ester (Calcein AM). Optimum adhe sive responsiveness occurred in bovine serum albumin (BSA)coated plates aft er 30 min incubation at 37 degrees C with 5 x 10(5) PMN in 100 mu l per wel l using phorbol myristate acetate (PMA, 100 ng/ml) as stimulant. The adhesi ve 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 (CVintra) for unstimulated and stimulated ad hesion in the assay was: CVintra = 12.9% and CVintra = 4.1%, respectively. The mean inter-assay coefficient of variation (CVinter) for unstimulated an d stimulated adhesion, respectively, in the assay was: CVinter = 22.9% and CVinter = 5.6%. The assay was highly reproducible and labelling with Calcei n AM neither reduced PMN viability nor activated the PMN. (C) 1999 Elsevier Science B.V. All rights reserved.