%0 Journal Article %A Düssmann, Heiko %A Perez-Alvarez, Sergio %A Anilkumar, Ujval %A Papkovsky, Dmitri B %A Prehn, Jochen HM %D 2019 %T Single-cell time-lapse imaging of intracellular O2 in response to metabolic inhibition and mitochondrial cytochrome-c release %U 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 %2 https://repository.rcsi.com/ndownloader/files/19301978 %K Antimycin A %K Cytochromes c %K Electron Transport Complex III %K Electron Transport Complex IV %K Fluorenes %K Gene Expression %K Genes %K Reporter %K Green Fluorescent Proteins %K HeLa Cells %K Humans %K Membrane Potential %K Mitochondrial %K Metalloporphyrins %K Mitochondria %K Molecular Probes %K Oxygen %K Polymers %K Rhodamines %K Single-Cell Analysis %K Sodium Azide %K Time-Lapse Imaging %K Physiology %K Medical Physics %X

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.

%I Royal College of Surgeons in Ireland