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Intracellular Oxygen Concentration Assay (ab197245)

Key features and details

  • Assay type: Quantitative
  • Detection method: Fluorescent
  • Platform: Microplate reader
  • Assay time: 1 hr 30 min
  • Sample type: Adherent cells, Suspension cells

Overview

  • Product name

    Intracellular Oxygen Concentration Assay

  • Detection method

    Fluorescent

  • Sample type

    Adherent cells, Suspension cells

  • Assay type

    Quantitative

  • Assay time

    1h 30m

  • Species reactivity

    Reacts with: Human
    Predicted to work with: Mammals

  • Product overview

    Intracellular Oxygen Concentration Assay (ab197245) is an easy mix and measure 96 well fluorescence plate reader based approach for the analysis of intracellular oxygen concentration at the cell monolayer. The assay is based on the ability of oxygen to quench the excited state of the oxygen-sensitive probe. The probe is taken up via nonspecific energy dependent endocytosis and, after washing, the cells are monitored on a fluorescence plate reader (dual-read TR-F required for full oxygen quantitation). The probe phosphorescence is quenched by intracellular oxygen in a non-chemical, reversible manner allowing the measurement of average intracellular O2 levels and facilitating real-time monitoring of relative changes in cellular oxygen consumption. The probe signal increases with a reduction in intracellular oxygen and deceases with an increase in intracellular oxygen. The probe is excitable at 360-400 or 535 nm and emits at 630-680 nm. Optimal filter combinations are Ex/Em = 340/642 nm.


    The flexible plate reader format, allows multiparametric or multiplex combination with a range of other reagents and it is suitable for HTP automation.


     

  • Notes

    Learn more about the full range of assays to measure glycolysis, oxygen consumption, fatty acid oxidation and metabolic flux in live cells.

    Or review the full metabolism assay guide for other assays for metabolites, metabolic enzymes, mitochondrial function, and oxidative stress.

  • Platform

    Microplate reader

Images

  • Excitation and emission spectra of O2 probe
    Excitation and emission spectra of O2 probe

    Excitation and Emission spectra of Intracellular O2 probe, showing normalized excitation (Ex 360-400nm; Peak 380nm) and emission (Em 630-670nm; Peak 650nm).

  • Illustrating dual read TR-F measurement.
    Illustrating dual read TR-F measurement.
  • HEK293T cell oxygenation
    HEK293T cell oxygenation

    HEK293T cell oxygenation. HEK293T cells were cultured in 2D and measured at ambient oxygen. Intracellular O2 levels were ~ 14%. Reducing instrument O2 to 6% caused cellular oxygenation to drop to ~2%. Assay performed using a CLARIOstart equipped with an ACU module (BMG Labtech).

  • Sample callibration data.
    Sample callibration data.

    Sample Calibration Data. Intra O2 probe Lifetime profiles measured at decreasing [O2] with parallel glucose oxidase treatment to achieve 0% O2.

  • Relationship between probe lifetime (t) and applied [O2].
    Relationship between probe lifetime (t) and applied [O2].

    Relationship between probe lifetime (τ) and applied [O2]. Applying a first order exponential fit generates a calibration function of O2% = A1 x Exp(-τ / t1). Example: O2% = 659.3 x Exp(-τ / 8.475).

  • Measuring the impact of cell metabolism on iPS-derived cardiomyocyte oxygenation.
    Measuring the impact of cell metabolism on iPS-derived cardiomyocyte oxygenation.

    Measuring the impact of cell metabolism on iPS-derived cardiomyocyte oxygenation. During measurement, cells are treated with antimycin (ETC inhibitor) and isoproterenol (β-adrenoreceptor agonist).

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