Submitted to: National Cytometry Symposium
Publication Type: Abstract Only
Publication Acceptance Date: December 14, 1997
Publication Date: N/A
Technical Abstract: Our laboratories are using flow cytometry for the study of neutrophil function, neutrophil cell surface receptors, and for sorting X and Y sperm. By using FITC-labeled bacteria, phagocytosis is measuerd after quenching adhered bacteria with methylene blue. Oxidative burst activity (hydrogen peroxide formation) involves measuring the oxidation of intracellular dichlorofluorescin to fluorescent dichlorofluorescein. Phorbol myristate acetate is used to perturb the neutrophil plasma membrane. The simultaneous measuremet of phagocytosis and oxidative burst activity can be accomplished by using propidium iodide stained bacteria and dihydrorhodamine 123, and processing the samples with the Coulter EPICS Q-Prep Immunology Workstation. We have produced a number of anti-bovine neutrophil MAB which cause neutrophils to undergo oxidative burst activity. These MAB together with MAB to bacteria were chemially linked to form bi-specific antibodies for targeted lysis of mastitis pathogens. Flow cytometric sperm sorting based on DNA is now a standardized method for sexing sperm. DNA is 3 to 4% greater in the X chromosome than in the Y chromosome. Hoeschst 33342, a DNA binding stain is added to intact viable sperm to differentiate X from Y sperm. Skewing of the sex ratio from the standard 50:50 to 85 to 90% in either the X or Y direction is effective in cattle, pigs, sheep, and rabbits. The sexing technology is an effective means for shifting the sex ratio towards the sex of greatest economic benefit to the livestock industry.