Resazurin reduction as a function of respiratory burst in bovine neutrophils.
OBJECTIVE
To determine whether the respiratory burst of neutrophils from bovine blood and milk can be analyzed by use of a fluorometric resazurin reduction assay.


METHODS
Neutrophils were obtained from EDTA-anticoagulated blood of 7 dairy cows. Neutrophils also were isolated from milk samples of a cow intramammarily challenge exposed with Escherichia coli lipopolysaccharide.


METHODS
The respiratory burst of neutrophils was analyzed in parallel, using the conventional luminol-enhanced luminometric procedure and a novel fluorometric procedure with resazurin as the fluorogenic substrate. Opsonized zymosan and phorbol myristate acetate were used as stimulants. The mechanism of the fluorescent response was analyzed, using metabolic inhibitors to various cell functions. Luminometry and fluorometry were carried out in parallel, using microtitration tray-reading instruments.


RESULTS
Stimulation of neutrophils induced resazurin reduction to resorufin and a fluorescent response. The luminescent response was transient, but the fluorescent response (build-up of fluorescent resorufin) was cumulative. Therefore, a single end-point measurement can be used for the fluorometric assay.


CONCLUSIONS
The proposed fluorometric microtitration tray technology is simple and has a high throughput capacity. The fluorometric and luminometric assays seem to have similar potential in the analysis of phagocyte functions.
Fang
W
W
Department of Clinical Sciences, Faculty of Veterinary Medicine, University of Helsinki, Finland.
Myllys
V
V
Sandholm
M
M
D000818:Animals; D001386:Azides / Q000494:pharmacology; D001773:Blood Cells / Q000166:cytology; D002273:Carcinogens / Q000494:pharmacology; D002417:Cattle / Q000097:blood / Q000378:metabolism / Q000502:physiology*; D005033:Ethylmaleimide / Q000494:pharmacology; D005260:Female; D005470:Fluorometry / Q000662:veterinary; D007202:Indicators and Reagents / Q000378:metabolism; D008163:Luminescent Measurements; D008892:Milk / Q000166:cytology; D009153:Mutagens / Q000494:pharmacology; D009243:NAD / Q000378:metabolism / Q000502:physiology; D019255:NADPH Oxidases / Q000378:metabolism / Q000502:physiology; D009504:Neutrophils / Q000166:cytology / Q000378:metabolism / Q000502:physiology*; D010078:Oxazines / Q000378:metabolism*; D010084:Oxidation-Reduction; D011134:Polysaccharides / Q000494:pharmacology; D016897:Respiratory Burst / Q000502:physiology*; D019810:Sodium Azide; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014149:Tranquilizing Agents / Q000494:pharmacology; D014268:Trifluoperazine / Q000494:pharmacology; D014966:Xanthenes; D015054:Zymosan / Q000494:pharmacology
D001386:Azides; D002273:Carcinogens; D007202:Indicators and Reagents; D009153:Mutagens; D010078:Oxazines; D011134:Polysaccharides; D013439:Sulfhydryl Reagents; D014149:Tranquilizing Agents; D014966:Xanthenes; D009243:NAD; C005843:resazurin; D014268:Trifluoperazine; D015054:Zymosan; D019810:Sodium Azide; D019255:NADPH Oxidases; D013755:Tetradecanoylphorbol Acetate; D005033:Ethylmaleimide