flow citometry phagocytosis and oxidative burst in horses
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8/3/2019 Flow Citometry Phagocytosis and Oxidative Burst in Horses
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Simultaneous Flow Cytometric Analysis of Phagocytosisand Oxidative Burst Activity in Equine Leukocytes
M.J.B.F. Flaminio1*, B.R. Rush2, E.G. Davis2, K. Hennessy3, W. Shuman4 and
M.J. Wilkerson4
1The James A. Baker Institute for Animal Health, College of Veterinary Medicine,
Cornell University, Hungerford Hill Road, Ithaca, New York 14853; 2Department of
Clinical Sciences, College of Veterinary Medicine, Kansas State University, Manhattan,
Kansas; 3Bayer Corporation, Agriculture and Animal Health Division, Merriam, Kansas;4Department of Diagnostic Medicine and Pathobiology, College of Veterinary Medicine,
Kansas State University, Manhattan, Kansas, USA*Correspondence: E-mail: [email protected]
Flaminio, M.J.B.F., Rush, B.R., Davis, E.G., Hennessy, K., Shuman, W. and Wilkerson, M.J., 2002.Simultaneous £ow cytometric analysis of phagocytosis and oxidative burst activity in equine leukocytes.Veterinary Research Communications, 26(2), 85^92
ABSTRACT
This paper describes a method for simultaneously measuring phagocytosis and oxidative burst activityin equine peripheral blood leukocytes by £ow cytometry. Opsonized propidium iodide-labelledStaphylococcus aureus (PI-Sa) was used to measure the uptake of bacteria by equine phacocytes andthe oxidative burst activity by oxidation of dihydrorhodamine 123. The requirements to achieveoptimal activity of phagocytosis and oxidative burst are described. The advantage of the simultaneous
technique is that it provides both independent and comparative values for phagocytosis and theoxidative burst, for the detection of impaired mechanisms of microbial destruction. Furthermore, thetechnique allows evaluation of opsonization activity in this context.
Keywords: £ow cytometry, horse, leukocytes, phagocytosis, propidium iodide, oxidative burst,rhodamine
Abbreviations: BAL, bronchoalveolar lavage £uid; DHR, dihydrorhodamine 123; FCS, forward lightscatter; HBSS, Hanks balanced salt solution; OBA, oxidative burst activity; PI, propidium iodide; PI-Sa, propidium-iodide-labelled Staphylococcus aureus; PMA, phorbol myristate acetate; SSC, side lightscatter; RHO, rhodamine 123; ROI, reactive oxygen intermediates
INTRODUCTION
We describe a method for simultaneously measuring phagocytosis and oxidative burstactivity (OBA) in equine peripheral blood leukocytes by £ow cytometry. Propidium
iodide-labelled Staphylococcus aureus (PI-Sa) was used to measure the uptake of
bacteria by equine phagocytes. Propidium iodide-stained S. aureus is rapidly pro-
cessed, is accessible and functions physiologically to stimulate an oxidative burst when
phagocytosed (BassÖe and Solberg, 1984). Trypan blue is necessary to quench the
£uorescent signals imparted by nonphagocytosed bacteria or extracellular bacteria
Veterinary Research Communications, 26 (2002) 85^92# 2002 Kluwer Academic Publishers. Printed in the Netherlands
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