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 after 30 min incubation at 37 degrees C with 5 x 10(5) PMN in 100 microl 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 (CVintra) for unstimulated and stimulated adhesion in the assay was: CVintra = 12.9% and CVintra = 4.1%, respectively. The mean inter-assay coefficient of variation (CVinter) for unstimulated and stimulated adhesion, respectively, in the assay was: CVinter = 22.9% and CVinter = 5.6%. The assay was highly reproducible and labelling with Calcein AM neither reduced PMN viability nor activated the PMN.
Petersen
T K
TK
Department of Clinical Studies, The Royal Veterinary and Agricultural University, Frederiksberg, Denmark. tkp@kvl.dk
Bysted
B V
BV
Jensen
A L
AL
D000268:Adhesiveness; D000818:Animals; D002448:Cell Adhesion; D004285:Dogs / Q000097:blood*; D005260:Female; D005452:Fluoresceins / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism; D005470:Fluorometry / Q000662:veterinary; D007958:Leukocyte Count / Q000662:veterinary; D008297:Male; D018375:Neutrophil Activation / Q000187:drug effects / Q000502:physiology; D009504:Neutrophils / Q000378:metabolism*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D005452:Fluoresceins; D005456:Fluorescent Dyes; C085925:calcein AM; D013755:Tetradecanoylphorbol Acetate