Flow cytometric detection of hyper-polarized mitochondria in regulated and accidental cell death processes
AbstractShikonin induced necroptosis in Jurkat cells were identified flow cytometrically by the up-regulation of RIP3 in live cells and that a proportion of these cells underwent other forms of regulated cell death (RCD) which included parthanatos (< 10%), or cleaved PARP (< 10%) and DNA Damage (> 30%). Live necroptotic cells also possessed functioning mitochondria with hyper-polarized mitochondria membrane potential and generated a fivefold increase in cellular reactive oxygen species (ROS) which was resistant to inhibition by zVAD and necrostatin-1 (Nec-1). After loss of plasma membrane integrity these dead necroptotic cells then showed a higher incidence of parthanatos (> 40%), or cleaved PARP (> 15%) but less DNA Damage (< 15%). Inhibition of shikonin induced apoptosis and necroptosis by zVAD and Nec-1 respectively resulted in live necroptotic cells with an increased incidence of cleaved PARP and reduced levels of DNA Damage respectively. Dead necroptotic cells then showed a reduced incidence of parthanatos and DNA Damage after inhibition by zVAD and Nec-1 respectively. A high proportion of these dead necroptotic cells (30%) which lacked plasma membrane integrity also displayed functioning hyper-polarized mitochondria with high levels of cellular ROS and thus had the capacity to influence the outcome of RCD p rocesses rather than just been the end product of cell death, the necrotic cell. Flow cytometry can thus measure multiple forms...
Source: Apoptosis - Category: Molecular Biology Source Type: research
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