Related Experiment Video
Updated: Oct 19, 2026
Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions
Published on: August 23, 2013
Determination of the adhesive capability of canine polymorphonuclear neutrophil granulocytes using a fluorometric
T K Petersen1, B V Bysted, A L Jensen
1Department of Clinical Studies, The Royal Veterinary and Agricultural University, Frederiksberg, Denmark. tkp@kvl.dk
Abstract:
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.
Insights
This study presents a rapid, reproducible fluorometric assay for measuring canine neutrophil adhesion. The assay uses calcein acetoxymethyl ester (Calcein AM) and is suitable for evaluating neutrophil function in laboratory settings.
Area of Science:
- Veterinary Immunology
- Cellular Assays
- Biotechnology
Background:
- Canine neutrophil function is crucial for immune response.
- Accurate and reproducible assays are needed to assess neutrophil adhesion.
- Existing methods may be time-consuming or lack sensitivity.
Purpose of the Study:
- To develop and validate a rapid fluorometric assay for canine polymorphonuclear neutrophil (PMN) adhesion.
- To optimize assay conditions for maximum sensitivity and reproducibility.
- To assess the reliability of the assay for use with laboratory beagles.
Main Methods:
- Evaluation of a fluorometric cellular adhesion assay using calcein acetoxymethyl ester (Calcein AM) in microtiter plates.
- Optimization of incubation time, temperature, cell concentration, and stimulant (phorbol myristate acetate - PMA).
- Determination of adhesive responsiveness in 15 laboratory beagle dogs and calculation of intra- and inter-assay coefficients of variation (CV).
Main Results:
- Optimal conditions involved bovine serum albumin (BSA)-coated plates, 30 min incubation at 37°C, 5 x 10^5 PMN/well, and PMA stimulation.
- Mean adherence for unstimulated PMN was 2.9% and for stimulated PMN was 74.3%.
- The assay demonstrated high reproducibility with low intra-assay (CVintra: 12.9% unstimulated, 4.1% stimulated) and inter-assay (CVinter: 22.9% unstimulated, 5.6% stimulated) coefficients of variation.
Conclusions:
- The developed fluorometric assay is rapid, reproducible, and reliable for quantifying canine PMN adhesion.
- Calcein AM labeling does not compromise PMN viability or induce activation, ensuring assay integrity.
- This assay provides a valuable tool for research and diagnostics involving canine neutrophil function.
