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Kinetic analysis of local oxygenation and respiratory responses of mammalian cells using intracellular oxygen-sensitive probes and time-resolved fluorometry

(2014) Methods in Enzymology. 542. p.183-207
Author
Organization
Abstract
The cell-penetrating O-2-sensing probes based on phosphorescent Pt-porphyrins provide a means to accurately measure O-2 within cultured cells in a contact-less fashion. Via this approach, in situ oxygenation and respiratory responses can be monitored in real time on large number of cellular samples seeded in standard microtiter plates, by means of a commercial time-resolved fluorescence reader. Using neuronal PC12 cells as a model, here we describe the general strategy behind the use of phosphorescent Pt-porphyrins to measure O-2 within cultured cells as well as detailed protocols to implement this experimental approach, including cell preparation and probe loading, instrument and plate setup, system calibration, and data processing. Two main protocols are presented: (1) a basic setup and optimization for the measurement of intracellular O-2 under ambient O-2 concentration; (2) the calibration of the system required to perform measurements under low O-2 concentrations (hypoxia). A case study on the effects of bafilomycin A1 on PC12 cell function is included, illustrating how these methods can be combined with conventional bioenergetic markers to delineate how chemical and/or physical stimuli affect the metabolism of normal and malignant cells.
Keywords
PC12 CELLS, MITOCHONDRIAL DYSFUNCTION, CEREBRAL-ISCHEMIA, CELLULAR, OXYGEN, O-2, FLUORESCENCE, HYPOXIA, BIOENERGETICS, COMPLEXES, PEPTIDES

Citation

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MLA
Zhdanov, Alexander V., et al. “Kinetic Analysis of Local Oxygenation and Respiratory Responses of Mammalian Cells Using Intracellular Oxygen-Sensitive Probes and Time-Resolved Fluorometry.” Methods in Enzymology, vol. 542, 2014, pp. 183–207, doi:10.1016/B978-0-12-416618-9.00010-8.
APA
Zhdanov, A. V., Dmitriev, R. I., Hynes, J., & Papkovsky, D. B. (2014). Kinetic analysis of local oxygenation and respiratory responses of mammalian cells using intracellular oxygen-sensitive probes and time-resolved fluorometry. Methods in Enzymology, 542, 183–207. https://doi.org/10.1016/B978-0-12-416618-9.00010-8
Chicago author-date
Zhdanov, Alexander V., Ruslan I. Dmitriev, James Hynes, and Dmitri B. Papkovsky. 2014. “Kinetic Analysis of Local Oxygenation and Respiratory Responses of Mammalian Cells Using Intracellular Oxygen-Sensitive Probes and Time-Resolved Fluorometry.” Methods in Enzymology 542: 183–207. https://doi.org/10.1016/B978-0-12-416618-9.00010-8.
Chicago author-date (all authors)
Zhdanov, Alexander V., Ruslan I. Dmitriev, James Hynes, and Dmitri B. Papkovsky. 2014. “Kinetic Analysis of Local Oxygenation and Respiratory Responses of Mammalian Cells Using Intracellular Oxygen-Sensitive Probes and Time-Resolved Fluorometry.” Methods in Enzymology 542: 183–207. doi:10.1016/B978-0-12-416618-9.00010-8.
Vancouver
1.
Zhdanov AV, Dmitriev RI, Hynes J, Papkovsky DB. Kinetic analysis of local oxygenation and respiratory responses of mammalian cells using intracellular oxygen-sensitive probes and time-resolved fluorometry. Methods in Enzymology. 2014;542:183–207.
IEEE
[1]
A. V. Zhdanov, R. I. Dmitriev, J. Hynes, and D. B. Papkovsky, “Kinetic analysis of local oxygenation and respiratory responses of mammalian cells using intracellular oxygen-sensitive probes and time-resolved fluorometry,” Methods in Enzymology, vol. 542, pp. 183–207, 2014.
@article{8687904,
  abstract     = {{The cell-penetrating O-2-sensing probes based on phosphorescent Pt-porphyrins provide a means to accurately measure O-2 within cultured cells in a contact-less fashion. Via this approach, in situ oxygenation and respiratory responses can be monitored in real time on large number of cellular samples seeded in standard microtiter plates, by means of a commercial time-resolved fluorescence reader. Using neuronal PC12 cells as a model, here we describe the general strategy behind the use of phosphorescent Pt-porphyrins to measure O-2 within cultured cells as well as detailed protocols to implement this experimental approach, including cell preparation and probe loading, instrument and plate setup, system calibration, and data processing. Two main protocols are presented: (1) a basic setup and optimization for the measurement of intracellular O-2 under ambient O-2 concentration; (2) the calibration of the system required to perform measurements under low O-2 concentrations (hypoxia). A case study on the effects of bafilomycin A1 on PC12 cell function is included, illustrating how these methods can be combined with conventional bioenergetic markers to delineate how chemical and/or physical stimuli affect the metabolism of normal and malignant cells.}},
  author       = {{Zhdanov, Alexander V. and Dmitriev, Ruslan I. and Hynes, James and Papkovsky, Dmitri B.}},
  isbn         = {{9780124166189}},
  issn         = {{0076-6879}},
  journal      = {{Methods in Enzymology}},
  keywords     = {{PC12 CELLS,MITOCHONDRIAL DYSFUNCTION,CEREBRAL-ISCHEMIA,CELLULAR,OXYGEN,O-2,FLUORESCENCE,HYPOXIA,BIOENERGETICS,COMPLEXES,PEPTIDES}},
  language     = {{eng}},
  pages        = {{183--207}},
  title        = {{Kinetic analysis of local oxygenation and respiratory responses of mammalian cells using intracellular oxygen-sensitive probes and time-resolved fluorometry}},
  url          = {{http://doi.org/10.1016/B978-0-12-416618-9.00010-8}},
  volume       = {{542}},
  year         = {{2014}},
}

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