Veterinary Research Communications, 26 (2002) 85^92 # 2002 Kluwer Academic Publishers.

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Simultaneous Flow Cytometric Analysis of Phagocytosis and 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 1 The 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; 4 Department of Diagnostic Medicine and Pathobiology, College of Veterinary Medicine, Kansas State University, Manhattan, Kansas, USA *Correspondence: E-mail: mbf6@cornell.edu
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 activity in equine peripheral blood leukocytes by ow cytometry. Opsonized propidium iodide-labelled Staphylococcus aureus (PI-Sa) was used to measure the uptake of bacteria by equine phacocytes and the oxidative burst activity by oxidation of dihydrorhodamine 123. The requirements to achieve optimal 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 the oxidative burst, for the detection of impaired mechanisms of microbial destruction. Furthermore, the technique 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 light scatter; HBSS, Hanks balanced salt solution; OBA, oxidative burst activity; PI, propidium iodide; PISa, propidium-iodide-labelled Staphylococcus aureus; PMA, phorbol myristate acetate; SSC, side light scatter; RHO, rhodamine 123; ROI, reactive oxygen intermediates

INTRODUCTION We describe a method for simultaneously measuring phagocytosis and oxidative burst activity (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 processed, is accessible and functions physiologically to stimulate an oxidative burst when phagocytosed (Basse and Solberg, 1984). Trypan blue is necessary to quench the uorescent signals imparted by nonphagocytosed bacteria or extracellular bacteria
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