Real‐time flow cytometry for the kinetic analysis of oncosis

Abstract

The standard method of distinguishing apoptotic and oncotic cells has been by microscopic analysis of nuclei and cell membrane morphology. Thus a rapid test for analyzing large numbers of cells in the study of cell necrobiology has not been possible until the recent advent of the Amnis Image-stream and real-time Lab-on-a-Chip technologies. An interesting difference between apoptosis and oncosis is that they are ATP dependent and independent processes, respectively. Here we describe an assay measuring real-time kinetic changes in the potential differences of the inner mitochondrial membrane (mmp) and the plasma membrane (pmp) in cells immediately before and after the addition of the inducing agent. Live cells were loaded with carbocyanine dye DiIC 1 (5) and bis-oxonol (DiBAC 4 (5)) to measure mmp and pmp in conjunction with annexin V-FITC and DAPI labeling for gating out annexin V binding cells and dead cells respectively. Live cells gave specific membrane signatures in response to apoptotic or oncotic reagents in real-time. Apoptosis showed little change in mmp and pmp signals over the course of 25 min, the mitochondria only showed a slight hyperpolarization. In contrast chemical treatment with oxidative phosphorylation blocker, sodium azide (SA) caused an immediate hyperpolarization spike followed by a complete abrogation of mmp over a 25 min time course. Treatment with SA (1%) also caused plasma membrane depolarization. Likewise detergent (0.01% Triton X-100) treatments also caused abrogation of mmp and depolarization of pmp. Whereas heat shock (428C) treatment showed only a slight mitochondrial membrane potential depolarization. These flow cytometric observations were confirmed by confocal microscopy. This novel real-time kinetic assay measuring mitochondrial and plasma membrane potential changes has important implications in the field of cell necrobiology in that it allows the researcher to differentiate apoptotic and oncotic processes in an immediate manner for the first time. '

Flow Cytometry Reveals the Nature of Oncotic Cells

“…The cell then undergoes osmosis and swelling with a bursting of cell organelles and the plasma membrane [11]. Uncoupling protein 2 (UCP-2), located in the inner mitochondrial membrane where protons are pumped by UCP-2 into the mitochondrial matrix or the intermembrane space, which then regulates ATP and superoxide production is modestly up-regulated by oncosis, resulting in a rapid depolarization of the mitochondrial membrane, which has been measured by flow cytometry [12,13]. The signal pathways involved in oncosis are under studied, so little information is known about these mechanisms compared to the knowledge of pathways in RCD [1,2,6].…”

“…Until recently, necrotic or oncotic cells were measured via flow cytometry using the Annexin V assay in which such cells are gated as cell viability +ve /AnnexinV –ve and numerous researchers have attempted to understand this dead cell population with varying success [13,15,16]. In our laboratory, mitochondrial and plasma membrane dysfunction were detected by the multiplexing of mitochondrial and plasma membrane probes into the Annexin V assay, leading to a better understanding of the biological processes occurring in this oncotic population [13,15,16]. The recent development of a polychromatic flow cytometric assay in this laboratory, which identifies most RCDs simultaneously and demonstrates pathways affected by use of pan-caspase and RIP1 protein blockers zVAD and necrostatin-1 (Nec-1), led us to re-investigate oncotic cell death for potential pathways by comparison with apoptosis.…”

MicroRNA Quantitation During Dendritic Cell Endocytosis Using Imaging Flow Cytometry: Key Factors and Requirements

“…This lack of accuracy is due to the low number of detected cells, with approximately 200-400 cells detected per field, though fluorescence microscopy is more accurate and quantitative in single cells [15]. Thus during key cellular events, subcellular information reflecting the cell population of interest is typically not accurate [16].…”

New insights into cell cycle and DNA damage response machineries through high‐resolution AMICO quantitative imaging cytometry

“…Further progress in research in these areas was thus advanced by the use of imaging cytometry utilizing either laser scanning cytometry (LSC) or image cytometry (3–10), flow imaging cytometry (11–13) or quantitative confocal microscopy (14,15). To expand capabilities of quantitative confocal microscopy in terms of analysis of proximity of sites of DNA damage induced by anticancer drugs vis-á-vis DNA replication sites (“replication factories”) further approaches have been recently developed utilizing computerized algorithms that allow to measure spatial immediacy of these sites in 3D space (16, 17).…”