In this application note we show how a MITO-ID® Intracellular O₂ Sensor Probe is performed on the CLARIOstar® microplate reader equipped with an atmospheric control unit (ACU). The ACU is a microprocessor-controlled unit that can regulate CO₂ and O₂ within the reader to reproduce the optimal physiological as well as hypoxic conditions needed for live cell-based assays. The MITO-ID® Intracellular O₂ Sensor nanoparticle probe is taken up by cells during an overnight loading period and responds in real time to any changes in intracellular oxygen concentration in both 2D culture as well as a wide range of 3D systems, including Matrigel, RAFT™, microtissues, Alvetex®, Mimetix® and other scaffolds. Oxygen quenches the phosphorescent emission from the probe, such that phosphorescence is proportional to [O₂].

Cellular respiration can reduce the levels of intracellular oxygen concentration, creating a local oxygen gradient. In the specific example shown here (Fig. 1) for cells cultured under ambient oxygen, the intracellular [O₂] measured using MITO-ID® Intracellular O₂ Sensor Probe was found to be ~14% for metabolically inactive cells and ~7% for metabolically active cell types. However, when the environmental O₂ concentration is reduced to ~6% using an ACU, the intracellular [O₂] falls to ~4.5% for metabolically less active cells, and is close to anoxia for metabolically active cells.

As the real oxygen concentration experienced by cells in culture is a function of environmental O₂ concentration, cell metabolism and seeding density, MITO-ID® Intracellular O₂ Sensor Probe provides the ideal tool to intelligently modulate these parameters to achieve a desired and specific intra-cellular oxygen concentration. Similarly, MITO-ID ® Intracellular O₂ Sensor Probe is an ideal tool to monitor real-time changes in intra-cellular [O₂] in response to treatments that perturb mitochondrial function and cell metabolism.