Repeatability of flow cytometric and classical measurement of phagocytosis and respiratory burst in bovine polymorphonuclear leukocytes.
Five methods for measurement of phagocytosis and respiratory burst activity of bovine blood polymorphonuclear leukocytes (PMNs) were evaluated. Eight cows were repeatedly sampled over a two week period and parallel samples tested in all five assays to assess the repeatability and stability of the methods. In the flow cytometric phagocytosis assay, ingestion of fluorescein labeled bacteria was measured, and in the flow cytometric assay for respiratory burst, oxidation of a dye by reactive oxygen species was recorded. In the classical assays, bactericidal effect on opsonized, live bacteria was quantified by the conversion of an indicator substance, superoxide anion production was assayed by the reduction of cytochrome c, whereas myeloperoxidase activity was determined with a radioactive iodination assay. The results showed that the Phagotest, Bursttest, cytochrome c and iodination assays gave repeatable results when samples were run in the same setup on the same day. Although day-to-day variability was significant in all assays, the described methods comprise a panel of useful tests for the evaluation of phagocytosis and respiratory burst activity in bovine PMNs. The flow cytometric methods represent a convenient alternative to the classical methods for measurement of phagocytosis and respiratory burst in bovine blood PMNs.
Kampen
Annette H
AH
National Veterinary Institute, P.O. Box 8156 Dep., N-0033 Oslo, Norway. annette.kampen@vetinst.no
Tollersrud
Tore
T
Larsen
Stig
S
Roth
James A
JA
Frank
Dagmar E
DE
Lund
Arve
A
D000818:Animals; D002417:Cattle / Q000097:blood / Q000276:immunology*; D004396:Coloring Agents / Q000494:pharmacology / Q000592:standards; D005260:Female; D005434:Flow Cytometry / Q000379:methods / Q000662:veterinary; D005562:Formazans / Q000494:pharmacology; D009504:Neutrophils / Q000276:immunology*; D009195:Peroxidase / Q000276:immunology; D010587:Phagocytosis / Q000276:immunology*; D011247:Pregnancy; D011933:Reagent Kits, Diagnostic / Q000662:veterinary; D015203:Reproducibility of Results; D016897:Respiratory Burst / Q000276:immunology*; D013481:Superoxides / Q000276:immunology; D013778:Tetrazolium Salts / Q000494:pharmacology
D004396:Coloring Agents; D005562:Formazans; D011933:Reagent Kits, Diagnostic; D013778:Tetrazolium Salts; D013481:Superoxides; C079782:MTT formazan; D009195:Peroxidase