Optimization of the under-agarose assay of porcine neutrophil migration

Glenn S. Elliott From the Department of Veterinary Pathobiology, School of Veterinary Medicine (Elliott, Rebar) and the Department of Statistics (McCabe, Alzola), Purdue University, West Lafayette, IN 47906.

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Alan H. Rebar From the Department of Veterinary Pathobiology, School of Veterinary Medicine (Elliott, Rebar) and the Department of Statistics (McCabe, Alzola), Purdue University, West Lafayette, IN 47906.

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George P. McCabe From the Department of Veterinary Pathobiology, School of Veterinary Medicine (Elliott, Rebar) and the Department of Statistics (McCabe, Alzola), Purdue University, West Lafayette, IN 47906.

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Carlos Alzola From the Department of Veterinary Pathobiology, School of Veterinary Medicine (Elliott, Rebar) and the Department of Statistics (McCabe, Alzola), Purdue University, West Lafayette, IN 47906.

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SUMMARY

Important procedural factors in the under-agarose assay for porcine neutrophil migration were identified, and optimal conditions were established. Three factors were tested: the concentration of zymosan-activated serum inoculated into the outer well; the number of neutrophils inoculated into the center well; and the time of incubation of the agarose plates. All factors had a significant (P < 0.0001, 0.0001, and 0.01, respectively) effect on the chemotactic index of porcine neutrophils. The optimal combination of these 3 factors was undiluted zymosan-activated serum as the chemoattractant, 8 × 105 neutrophils inoculated into the center well, and 5 hours of incubation. The assay was validated, using standard conditions, and the data were used to predict the number of pigs and/or repetitive assays needed to identify differences among experimental groups.

SUMMARY

Important procedural factors in the under-agarose assay for porcine neutrophil migration were identified, and optimal conditions were established. Three factors were tested: the concentration of zymosan-activated serum inoculated into the outer well; the number of neutrophils inoculated into the center well; and the time of incubation of the agarose plates. All factors had a significant (P < 0.0001, 0.0001, and 0.01, respectively) effect on the chemotactic index of porcine neutrophils. The optimal combination of these 3 factors was undiluted zymosan-activated serum as the chemoattractant, 8 × 105 neutrophils inoculated into the center well, and 5 hours of incubation. The assay was validated, using standard conditions, and the data were used to predict the number of pigs and/or repetitive assays needed to identify differences among experimental groups.

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