Intracellular Oxygen Concentration Assay (ab197245)

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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).

References (3)

Publishing research using ab197245? Please let us know so that we can cite the reference in this datasheet.

ab197245 has been referenced in 3 publications.

  • Uribe-Alvarez C  et al. Wolbachia pipientis grows in Saccharomyces cerevisiae evoking early death of the host and deregulation of mitochondrial metabolism. Microbiologyopen 8:e00675 (2019). PubMed: 29897678
  • Cubillos-Zapata C  et al. Obstructive Sleep Apnea Monocytes Exhibit High Levels of Vascular Endothelial Growth Factor Secretion, Augmenting Tumor Progression. Mediators Inflamm 2018:7373921 (2018). PubMed: 29997451
  • Hernández-Jiménez E  et al. Monocytes inhibit NK activity via TGF-ß in patients with obstructive sleep apnoea. Eur Respir J 49:N/A (2017). PubMed: 28619958

Customer reviews and Q&As

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Question

Inquiry: Is the Intracellular Oxygen Concentration Assay (Cat No 197245) compatible with flow cytometry? Thanks.

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Answer

This product is not recommended for use in flow cytometry, however in theory it would be possible to detect the reagent given the correct excitation and emission and measurement of reagent loaded cells. However the assay set-up and conditions would likely not be suitable to allow detectable changes in intracellular O2, to be measured sensitivity enough on that instrument as the assay format is designed for plate reader analysis of a 2D monolayer or a 3D structure containing reagent loaded cells.

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