Bigdelou Parnian, Farnoud Amir M
Biomedical Engineering Program, Ohio University.
Biomedical Engineering Program, Ohio University; Department of Chemical and Biomolecular Engineering, Ohio University;
J Vis Exp. 2020 Jan 21(155). doi: 10.3791/60659.
Eryptosis, erythrocyte programmed cell death, occurs in a number of hematological diseases and during injury to erythrocytes. A hallmark of eryptotic cells is the loss of compositional asymmetry of the cell membrane, leading to the translocation of phosphatidylserine to the membrane outer leaflet. This process is triggered by increased intracellular concentration of Ca, which activates scramblase, an enzyme that facilitates bidirectional movement of phospholipids between membrane leaflets. Given the importance of eryptosis in various diseased conditions, there have been efforts to induce eryptosis in vitro. Such efforts have generally relied on the calcium ionophore, ionomycin, to enhance intracellular Ca concentration and induce eryptosis. However, many discrepancies have been reported in the literature regarding the procedure for inducing eryptosis using ionomycin. Herein, we report a step-by-step protocol for ionomycin-induced eryptosis in human erythrocytes. We focus on important steps in the procedure including the ionophore concentration, incubation time, and glucose depletion, and provide representative result. This protocol can be used to reproducibly induce eryptosis in the laboratory.
红细胞程序性死亡（eryptosis）发生于多种血液系统疾病以及红细胞受到损伤的过程中。凋亡细胞的一个标志是细胞膜成分不对称性的丧失，导致磷脂酰丝氨酸易位至细胞膜外小叶。这个过程由细胞内钙离子浓度升高触发，钙离子激活了翻转酶，该酶促进磷脂在细胞膜小叶间的双向移动。鉴于红细胞程序性死亡在各种疾病状态中的重要性，人们一直在努力在体外诱导红细胞程序性死亡。此类努力通常依赖钙离子载体离子霉素来提高细胞内钙离子浓度并诱导红细胞程序性死亡。然而，文献中报道了许多关于使用离子霉素诱导红细胞程序性死亡的方法存在差异。在此，我们报告一种在人红细胞中使用离子霉素诱导红细胞程序性死亡的分步方案。我们关注该过程中的重要步骤，包括离子载体浓度、孵育时间和葡萄糖耗竭，并提供代表性结果。该方案可用于在实验室中可重复地诱导红细胞程序性死亡。
