Cytolytic peptides induce biphasic permeability changes in mammalian cell membranes
Cytolytic peptides induce biphasic permeability changes in mammalian cell membranes
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DOI：
10.1016/s0022-1759(01)00334-9
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发表时间：
2001-06-01
影响因子：
2.2
通讯作者：
Mentzer, SJ
中科院分区：
文献类型：
--
作者：
Su, M;He, CF;Mentzer, SJ
溶细胞肽蜂毒肽和短杆菌肽S是天然存在的试剂，为补体、免疫毒素和细胞介导的膜通透性的研究提供了比较模型。大多数表征溶细胞肽的尝试都使用模型膜系统，包括磷脂囊泡或红细胞。膜囊泡允许使用荧光渗透性标记物的自猝灭浓度。而红细胞释放可测量的血红蛋白。测量有核哺乳动物细胞早期膜通透性变化的尝试受到限制。为了测量由细胞溶解肽诱导的哺乳动物细胞膜通透性变化的动力学，我们开发了使用细胞质荧光染料钙黄绿素作为膜通透性标记物的96孔荧光细胞溶解测定法。为了促进膜渗透性的快速评估，将台盼蓝加入到测定溶液中以淬灭（a）释放的荧光和（B）保留的细胞内荧光。台盼蓝还提供了细胞活力的补充目视评估。使用该测定，详细的动力学分析证明了细胞膜在暴露于溶细胞肽的数秒内的渗透性。使用钙指示剂染料fluo-3通过流式细胞术证实细胞膜的快速透化。该测定还证明了在接下来的几个小时内标志物释放的第二个较慢阶段。荧光细胞溶解试验能够可靠地检测与蜂毒肽和短杆菌肽S相关的双相渗透性变化，这表明该试验在评估其他细胞溶解剂中的潜在实用性。(C)2001 Elsevier Science B. V.保留所有权利。
The cytolytic peptides melittin and gramicidin S are naturally occurring agents that provide a comparative model for studies of complement, immunotoxin and cell-mediated membrane permeability. Most attempts to characterize cytolytic peptides have used model membrane systems including phospholipid vesicles or erythrocytes. Membrane vesicles permit the use of self-quenching concentrations of fluorescent permeability markers. while erythrocytes release measurable hemoglobin. Attempts at measuring early membrane permeability changes in nucleated mammalian cells have been limited. To measure the kinetics of mammalian cell membrane permeability changes induced by cytolytic peptides, we developed a 96-well fluorescence cytolysis assay using the cytoplasmic fluorescent dye calcein as the membrane permeability marker. To facilitate rapid assessment of membrane permeability, trypan blue was added to the assay solution to quench (a) released fluorescence and (b) retained intracellular fluorescence. Trypan blue also provided a complementary visual assessment of cell viability. Using this assay, a detailed kinetic analysis demonstrated permeability of the cell membranes within seconds of exposure to the cytolytic peptides. The rapid permeabilization of the cell membranes was confirmed by flow cytometry using the calcium indicator dye fluo-3, The assay also demonstrated a second slower phase of marker release over the next several hours. The fluorescence cytolysis assay was able to reliably detect the biphasic permeability changes associated with the melittin and gramicidin S peptides suggesting the potential utility of this assay in the assessment of other cytolytic agents. (C) 2001 Elsevier Science B.V. All rights reserved.