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

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.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.A