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KMID : 1023519990220040377
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Korean Journal of Veterinary Service
1999 Volume.22 No. 4 p.377 ~ p.383
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Functional studies of granulocytes in ruminants 1. Rapid separation of polymorphonuclear leucocytes from circulating blood in bovine
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Park Il-Kyu
Yoon Chang-Yong
Lee Jeoung-Won
Song Hee-Jong
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Abstract
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Polymorphonuclear (PMN) leucocytes are fundamental importance to the body’s defense mechanism and play a major role in the local and systemic reactions to infectious disease. Investigation of the physiological and pathological role of the various leucocyte subtypes in host defence mechanisms is dependent upon the isolation of adequate numbers of viable, pure leucocyte fractions. This report describes the separate frequency of PMN leucocytes both from buffy coat layer and from packed RBC layer when bovine peripheral blood was treated with various anti-coagulants such as acid-citrate-dextrose(ACD), ethyldiaminetetraacetic acid(EDTA), sodium citrate and heparin. The separate frequencies of PMN leucocytes from buffy coat layer was 60.49.6%(heparin), 56.811.8%(sodium citrate), 30.614.1%(ACD) and 6.23.7%(EDTA), in order. Those from packed RBC layer monitored with EDTA, ACD, sodium citrate and heparin was 85.04.7%, 84.35.5%, 83.86.5% and 76.37.7%, respectively. The Ficoll-hypaque(FH) density gradient method was used to remove a small part of lymphocytes and/or monocytes from leucocytes in packed RBC layer. With the result that it increased separate frequency of PMN leucocytes from EDTA(89.92.4%), ACD(89.53.6%), and sodium citrate(83.610.3%) than heparin(68.413.9%). These results indicate that the use of EDTA and ACD as anticoagulant Is suitable for the separation of PMN leucocytes from bovine peripheral blood, and that the FH density gradient method is able to increase the separate frequency of PMN leucocytes from packed RBC layer.
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KEYWORD
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Bovine, PMN leucocyte isolation, Anticoagulants, Centrifugation
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