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Real-time Measurement of Intracellular O2 in Mammalian Cells

  • New O2-sensitive cell-penetrating nanoparticle probe MITO-ID® Intracellular Oxygen Assay
  • Cytosolic O2 tension significantly influences signal transduction and cellular metabolism
  • Dedicated measurement protocols, [O2] scale conversion and data analysis with one mouse click

Franka Maurer, BMG LABTECH, Germany
Wini Luty, Enzo Life Sciences, Farmingdale, USA

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MITO-ID® Intracellular O2 Sensor Probe


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Introduction & Assay Principle

As illustrated in recent publications, the level of available oxygen in the cell significantly influences cell physiology, signal transduction and cellular response to drug treatment. In spite of this knowledge, the majority of in vitro studies culture and study cells at ambient oxygen – ignoring the oxygen gradient between the atmosphere and the medium and between the medium and the intracellular cell environment. To facilitate the quantification of cellular oxygenation Enzo Life Sciences has developed the MITO-ID® Intracellular O2 Sensor Probe, based on a proprietary O2-sensitive cell-penetrating nanoparticle probe.

Fig. 1: (A) Schematic illustration showing changing O2 tension in tissues. (B) In vitro cell culture environmental [O2] set to ~6% using an ACU and (C) corresponding cellular [O2] measured by MITO-ID® Intracellular O2 Sensor Probe for metabolically active (blue) and metabolically less active cells (red).

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.

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