原创力文档application of flow cytometry to determine differential redistribution of cytochrome c and smacdiablo from mitochondria during cell death signaling应用流式细胞仪来确定微分再分配的细胞色素c和smacdiablo线粒体在细胞死亡信号.pdfVIP
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application of flow cytometry to determine differential redistribution of cytochrome c and smacdiablo from mitochondria during cell death signaling应用流式细胞仪来确定微分再分配的细胞色素c和smacdiablo线粒体在细胞死亡信号
Application of Flow Cytometry to Determine Differential
Redistribution of Cytochrome c and Smac/DIABLO from
Mitochondria during Cell Death Signaling
Heling Ng, Danielle J. Smith, Phillip Nagley*
Department of Biochemistry and Molecular Biology, and ARC Centre of Excellence in Structural and Functional Microbial Genomics, Monash University, Clayton, Victoria,
Australia
Abstract
Mitochondrially mediated apoptosis is characterized by redistribution of proteins from mitochondria to cytoplasm following
permeabilization of the outer mitochondrial membrane. We applied flow cytometry to quantify simultaneously the
redistribution of two apoptogenic proteins, cytochrome c (cyt c) and Smac/DIABLO (Smac). Mammalian cells were treated
with digitonin that selectively permeabilizes the plasma membrane. Following fixation, treated cells were infused
successively with primary and secondary antibodies (the latter fluorescently tagged) enabling independent detection of cyt
c and Smac. Digitonin-treated cells that retain cyt c or Smac in mitochondria generate strong fluorescence signals in flow
cytometry. Cells in which cyt c or Smac have transited the outer mitochondrial membrane show greatly reduced
fluorescence because the proteins are lost from the digitonin-permeabilized cells. Quantitative flow cytometry revealed that
in 143B TK- cells treated with staurosporine, cyt c and Smac exit mitochondria asymmetrically, with cyt c redistribution
preceding that of Smac. However, in HeLa cells likewise treated, cyt c and Smac exit mitochondria concurrently. Under other
conditions of apoptotic induction, for example, 143B TK- cells treated with MT-21 (an apoptotic inducer that binds to the
mitochondrial adenine nucleotide transporter), redistribution of Smac prece
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