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

Abstract

The cell-penetrating O2-sensing probes based on phosphorescent Pt-porphyrins provide a means to accurately measure O2 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 O2 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 O2 under ambient O2 concentration; (2) the calibration of the system required to perform measurements under low O2 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: Hypoxia; Oxygen; Phosphorescence quenching; Pt-porphyrin probes; Respiration; Time-resolved fluorescence reader.