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Single-cell time-lapse imaging of intracellular O2 in response to metabolic inhibition and mitochondrial cytochrome-c release
Düssmann, Heiko; Perez-Alvarez, Sergio; Anilkumar, Ujval; Papkovsky, Dmitri B; Prehn, Jochen HM
<p>The original article is available at http://www.nature.com</p> <p>The detection of intracellular molecular oxygen (O2) levels is important for understanding cell physiology, cell death, and drug effects, and has recently been improved with the development of oxygen-sensitive probes that are compatible with live cell time-lapse microscopy. We here provide a protocol for the use of the nanoparticle probe MitoImage-MM2 to monitor intracellular oxygen levels by confocal microscopy under baseline conditions, in response to mitochondrial toxins, and following mitochondrial cytochrome-c release. We demonstrate that the MitoImage-MM2 probe, which embeds Pt(II)-5,10,15,20-tetrakis-(2,3,4,5,6-pentafluorophenyl)-porphyrin as oxygen sensor and poly(9,9-dioctylfluorene) as an O2-independent component, enables quantitative, ratiometric time-lapse imaging of intracellular O2. Multiplexing with tetra-methyl-rhodamine-methyl ester in HeLa cervical cancer cells showed significant increases in intracellular O2 accompanied by strong mitochondrial depolarization when respiratory chain complexes III or IV were inhibited by Antimycin A or sodium azide, respectively, and when cells were maintained at 'physiological' tissue O2 levels (5% O2). Multiplexing also allowed us to monitor intracellular O2 during the apoptotic signaling process of mitochondrial outer membrane permeabilization in HeLa expressing cytochrome-c-eGFP, and demonstrated that mitochondria post cytochrome-c release are able to retain their capacity to respire at physiological O2 despite a decrease in mitochondrial membrane potential.</p>
Keyword(s): Antimycin A; Cytochromes c; Electron Transport Complex III; Electron Transport Complex IV; Fluorenes; Gene Expression; Genes; Reporter; Green Fluorescent Proteins; HeLa Cells; Humans; Membrane Potential; Mitochondrial; Metalloporphyrins; Mitochondria; Molecular Probes; Oxygen; Polymers; Rhodamines; Single-Cell Analysis
Publication Date:
2017
Type: Journal article
Peer-Reviewed: Yes
Institution: Royal College of Surgeons in Ireland
Citation(s): Dussmann H, Perez-Alvarez S, Anilkumar U, Papkovsky DB, Prehn J. Single-cell time-lapse imaging of intracellular O2 in response to metabolic inhibition and mitochondrial cytochrome-c release. Cell Death & Disease. 2017;8(6)e2853
Publisher(s): Springer Nature
File Format(s): application/pdf
Related Link(s): https://epubs.rcsi.ie/physiolart/121
First Indexed: 2018-03-14 07:16:27 Last Updated: 2018-06-09 07:15:44