Isolation and characterization of mammalian cells that are undergoing apoptosis by a bovine serum albumin density gradient
Isolation and characterization of mammalian cells that are undergoing apoptosis by a bovine serum albumin density gradient
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DOI：
10.1016/j.ab.2004.05.035
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发表时间：
2004-09-15
影响因子：
2.9
通讯作者：
Nishi, Y
中科院分区：
文献类型：
--
作者：
Ajiro, K;Th'ng, J;Nishi, Y
当细胞被细胞毒剂处理时，它们不同步地进入凋亡，从而产生处于细胞退化的不同阶段的细胞。这种混合物使研究导致细胞死亡的生化途径变得困难。我们在一个简单的不连续的牛血清白蛋白(BSA)密度梯度离心法中将凋亡的哺乳动物细胞分成五层，每一层都包含处于不同阶段的细胞，(1)非凋亡的，(2)经历凋亡的，(3)成熟的凋亡细胞，根据染色质凝聚的光镜和电子显微镜以及DNA片段化的程度来判断。在这些正在发生凋亡的细胞中，包括c-jun氨基末端激酶/应激激活蛋白激酶和proaspase3裂解在内的凋亡标志物的修饰是明显的。在发生凋亡的细胞中，组蛋白H_2B的磷酸化水平显著升高，并在细胞质中观察到DNA片段化活性，而在其他组分的细胞中观察到的DNA断裂活性不高。结果表明，用BSA梯度法可以很容易地对凋亡细胞进行分级，该方法对于研究导致细胞凋亡的生化过程具有重要的价值。(C)2004 Elsevier Inc.保留所有权利。
When cells are treated with cytotoxic agents, they enter apoptosis asynchronously to yield cells at various stages of cellular deterioration. This mixture makes it difficult to Study the biochemical pathways leading to cell death. We have fractionated apoptotic mammalian cells in a simple discontinuous bovine serum albumin (BSA) density gradient centrifugation into five layers, each containing cells at different stages of apoptosis, (1) nonapoptotic, (2) undergoing apoptosis, and (3) mature apoptotic cells, as judged by light and electron microscopy of chromatin condensation and by the extent of DNA fragmentation. Modifications of apoptosis markers including c-Jun N-terminal kinase/stress-activated protein kinase and procaspase 3 cleavage were apparent in those cells that are undergoing apoptosis. Apoptosis-specific histone H2B phosphorylation was highly elevated and DNA fragmentation activity in the cytoplasm was observed in those cells that are undergoing apoptosis, but not much was observed in the cells of other fractions. Results show that apoptotic cells can be fractionated easily by the BSA gradient method, and this method will be invaluable for studying the biochemical processes that drive apoptosis. (C) 2004 Elsevier Inc. All rights reserved.