Koenderman L, Kok P T, Hamelink M L, Verhoeven A J, Bruijnzeel P L
Central Laboratory, Netherlands Red Cross Blood Transfusion Service, Amsterdam.
J Leukoc Biol. 1988 Aug;44(2):79-86. doi: 10.1002/jlb.44.2.79.
A simple and improved procedure is described for the isolation of human eosinophils from normal individuals with about 2% eosinophils in their peripheral blood. This method comprises a preincubation of a mixed granulocyte preparation with 10 nM fMLP for 10 min at 37 degrees C followed by a one-step density centrifugation on isotonic Percoll. The recovery of eosinophils is 49 +/- 4% at 89 +/- 4% purity. Because of the relatively high rate of recovery, it is now possible to isolate eosinophils from blood samples as small as 20 ml. Because treatment with fMLP may alter the functional activity of the eosinophils, the following metabolic functions were tested: changes in cytosolic free Ca2+, oxygen consumption, chemiluminescence, chemotaxis, and leukotriene C4 formation. We found that 10 nM fMLP does not activate eosinophils in these assays, whereas 1 microM fMLP does (with the exception of chemotaxis). Furthermore, pretreatment of eosinophils with 10 nM fMLP did not influence the response to other stimuli in these assays. The usefulness of this method was evaluated by comparing it with three other previously described procedures. In our hands, only the method presented here enabled us to isolate eosinophils from normal individuals with about 2% eosinophils in their peripheral blood.
本文描述了一种简单且改进的方法，用于从外周血中嗜酸性粒细胞约占2%的正常个体中分离人嗜酸性粒细胞。该方法包括将混合粒细胞制剂与10 nM fMLP在37℃下预孵育10分钟，然后在等渗 Percoll 上进行一步密度离心。嗜酸性粒细胞的回收率为49±4%，纯度为89±4%。由于回收率相对较高，现在可以从低至20 ml的血样中分离嗜酸性粒细胞。由于用fMLP处理可能会改变嗜酸性粒细胞的功能活性，因此测试了以下代谢功能：胞质游离Ca2+的变化、耗氧量、化学发光、趋化性和白三烯C4的形成。我们发现在这些测定中10 nM fMLP不会激活嗜酸性粒细胞，而1 μM fMLP会激活（趋化性除外）。此外，用10 nM fMLP对嗜酸性粒细胞进行预处理不会影响这些测定中对其他刺激的反应。通过将该方法与之前描述的其他三种方法进行比较，评估了该方法的实用性。在我们的操作中，只有本文介绍的方法能够让我们从外周血中嗜酸性粒细胞约占2%的正常个体中分离出嗜酸性粒细胞。
