Roth J A, Kaeberle M L
J Immunol Methods. 1981;45(2):153-64. doi: 10.1016/0022-1759(81)90209-x.
A procedure for isolating purified populations of neutrophils and eosinophils from bovine peripheral blood is described. The procedure involves the centrifugation of anti-coagulated whole bovine blood, flash lysis of the packed red blood cells and separation of the eosinophils from the neutrophils by centrifugation on a Ficoll-Hypaque density gradient. The following parameters were evaluated on populations of greater than 90% pure eosinophils and neutrophils: (1) random migration under agarose, (2) ingestion of 125I-labeled Staphylococcus aureus, (3) nitroblue tetrazolium reduction, (4) iodination, or the conversion of 125I to a trichloroacetic acid precipitable form, (5) antibody-dependent cell-mediated cytotoxicity, and (6) the amount of intracellular peroxidase. Neutrophils were significantly more active than eosinophils in the ingestion of S. aureus and in antibody-dependent cell-mediated cytotoxicity. Eosinophils were much more active than neutrophils in the resting and stimulated iodination test and they contained higher levels of peroxidase. There was no difference between the two cell types in their ability to reduce nitroblue tetrazolium or migrate under agarose. The effect of Ficoll-Hypaque on granulocyte function was determined. Previous contact with Ficoll-Hypaque significantly reduced random migration but other function tests were unaffected.
本文描述了一种从牛外周血中分离纯化嗜中性粒细胞和嗜酸性粒细胞群体的方法。该方法包括对抗凝的全牛血进行离心，对压实的红细胞进行快速裂解，并通过在Ficoll-Hypaque密度梯度上离心从中性粒细胞中分离嗜酸性粒细胞。对纯度大于90%的嗜酸性粒细胞和中性粒细胞群体评估了以下参数：(1) 在琼脂糖下的随机迁移，(2) 摄取125I标记的金黄色葡萄球菌，(3) 硝基蓝四氮唑还原，(4) 碘化，或将125I转化为三氯乙酸可沉淀形式，(5) 抗体依赖性细胞介导的细胞毒性，以及(6) 细胞内过氧化物酶的量。在摄取金黄色葡萄球菌和抗体依赖性细胞介导的细胞毒性方面，中性粒细胞比嗜酸性粒细胞明显更活跃。在静息和刺激碘化试验中，嗜酸性粒细胞比中性粒细胞活跃得多，并且它们含有更高水平的过氧化物酶。在还原硝基蓝四氮唑或在琼脂糖下迁移的能力方面，两种细胞类型之间没有差异。确定了Ficoll-Hypaque对粒细胞功能的影响。先前与Ficoll-Hypaque接触显著降低了随机迁移，但其他功能测试未受影响。
