Single-cell time-lapse imaging of intracellular O2 in response to metabolic inhibition and mitochondrial cytochrome-c release Heiko Düssmann Sergio Perez-Alvarez Ujval Anilkumar Dmitri B Papkovsky Jochen HM Prehn 10779/rcsi.10789508.v1 https://repository.rcsi.com/articles/journal_contribution/Single-cell_time-lapse_imaging_of_intracellular_O2_in_response_to_metabolic_inhibition_and_mitochondrial_cytochrome-c_release/10789508 <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> 2019-11-22 16:51:36 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 Sodium Azide Time-Lapse Imaging Physiology Medical Physics