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J Pharmacol Toxicol Methods. 1995 Apr;33(2):91-100.

Separation of neutrophils from blood in human and laboratory animals and comparison of the chemotaxis.

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Drug Safety Research Center, Daiichi Pharmaceutical Co., Ltd., Tokyo, Japan.


We separated neutrophils from the peripheral blood of the human, monkey, dog, rabbit, rat, hamster, and mouse and investigated their responses to several chemotactic factors to evaluate the species-dependent difference in responsiveness. Heparinized blood was obtained by venipuncture from human, monkey, dog, and rabbit. In the rat, hamster, and mouse, the hetastarch exchange transfusion method was used to increase the neutrophil recovery. After sedimentation of red blood cells, a leukocyte suspension was layered on 5-step discontinuous Percoll gradients of densities from 1.081 to 1.097. We could routinely obtain the fractions containing neutrophils at more than 90% in all of the seven species tested. Chemotaxis assay was performed using a 48-well modified Boyden chamber. Dog neutrophils did not migrate to N-formylmethionyl-leucyl-phenylalamine (fMLP), and dog and rat neutrophil responses to leukotriene B4 (LTB4) were remarkably modest. However, neutrophils from other species showed a high reactivity to fMLP and LTB4. Although neutrophils of all species responded to human recombinant interleukin 8 (hrIL-8), the sensitivity of human and monkey neutrophils to hrIL-8 were higher as compared with other species. Human, dog, and rat neutrophils reacted most to homologous zymosan-activated serum derived respectively from human, dog, and rat serum. The present results show species-dependent differences of neutrophils in chemotactic responses.

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