製品の概要

  • 製品名

    Extracellular Oxygen Consumption Assay
    Extracellular Oxygen Consumption キット 製品一覧
  • 検出方法

    Fluorescent
  • サンプルの種類

    Tissue, Adherent cells, Suspension cells, Purified mitochondria
  • アッセイタイプ

    Cell-based
  • 全工程の試験時間

    1h 30m
  • 製品の概要

    Extracellular Oxygen Consumption Assay Kit ab197243 is a mix-and-read, 96-well fluorescence plate reader assay for the real-time kinetic analysis of extracellular oxygen consumption rates (OCR). The oxygen consumption rate is a measure of the cellular respiration rate, and of mitochondrial function.


    The assay is optimized for isolated mitochondria and cell cultures, and can be used with tissues, enzyme preparations, and small organisms.


    The fluorescent dye used in this assay kit is quenched by oxygen. The dye excites at 360-380 nm (max 380) and emits at 630-680 nm (max 650). It is also available separately as ab197242.


    In the assay, an oil layer is added on top of the assay medium to limit diffusion of oxygen into the assay medium. As mitochondrial respiration depletes the oxygen within the assay medium, quenching of the fluorescent dye is reduced, and the fluorescence signal increases proportionately. 


    The reaction is non-destructive and fully reversible (the oxygen sensitive dye is not consumed) enabling assay time courses and drug treatments.

  • 特記事項

    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.

  • 試験プラットフォーム

    Microplate reader

製品の特性

画像

  • Cellular Energy Flux for HepG2 cells (seeded at 65,000 per well), treated with a combination of drug compounds modulating the ETC (Antimycin A [1 µM] and FCCP [2.5 µM]), shown as a percentage relative to untreated control cells. Comparative measurements were taken with Extracellular Oxygen Consumption Assay (ab197243) (white column) and Glycolysis Assay [Extracellular acidification] (ab197244) (black column) show the shift between mitochondrial respiration and glycolysis and the cellular control of energy (ATP; measured 1h post-treatment using Luminescent ATP Detection Assay kit (ab113849) (striped column)).

  • Typical Lifetime profile of Extracellular O2 Consumption Assay for adherent cells, treated with different ETC compounds, including Antimycin A (recommended as a Negative Control). The effect of Glucose Oxidase as a positive Signal Control is illustrated schematically.

  • Excitation and emission spectra of Extracellular O2 Consumption Reagent. Left panel shows normalized excitation (Ex = 360-400nm; Peak 380nm). Right panel shows emission (Em = 630 - 680nm; Peak 650nm) in oxygenated and deoxygenated conditions.

プロトコール

参考文献

This product has been referenced in:

  • Kang HM  et al. Body size-dependent interspecific tolerance to cadmium and their molecular responses in the marine rotifer Brachionus spp. Aquat Toxicol 206:195-202 (2019). Read more (PubMed: 30500606) »
  • Silao FGS  et al. Mitochondrial proline catabolism activates Ras1/cAMP/PKA-induced filamentation in Candida albicans. PLoS Genet 15:e1007976 (2019). Read more (PubMed: 30742618) »
See all 17 Publications for this product

レビューと Q&A

1-2 of 2 Abreviews or Q&A

Answer

Yes, you can use a TECAN F500 instrument, once it has fluorescence detection, suitable excitation and  wavelengths capabilities for MitoXpress ( i.e. 380nm, & 650nm), as well as temperature control. We would advise using single TR-F detection (standard measurement mode) if available and check you have a suitable filter for the right excitation and emission available. The Assay works well on many Tecan instruments, once proper parameters are employed.

Please note the instrument list does not prohibit use of any other fluorescence plate reader. The list contains the highest recommended readers and highest recommended measurement modes (Basic, Standard or Advanced), we advise. The measurement modes are listed in order of priority.

The kit has been used in the past with SH-SY5Y human neuroblastoma cell line. It is important to perform cell titration experiment to identify suitable seeding density for optimum detection in your individual samples.

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Answer

You can use a similar set up as the one described in the protocol, starting with the recommended dilution of 1:15 for a 96-wp. However, if tissue samples tested need to be larger, we would suggest that you adjust the assay for use in 48-wp or even 24-wp. In this case, however, you would have to do some asay optimization as it will consume a greater volume of reagents and mineral oil.

For instance, for 48-well plates, the assay volume would increase to 300ul - 400ul, therefore using 2X or more reagent, the assay volume for measurement needs a large enough to yield long enough path length to measure enough reliable signal from the well.

Tissue samples should be prepared in the same way as described in the protocol, but measurement should be conducted in the culture media used to culture the tissues, so the samples can metabolize and consume O2 as normal.

There is no need or benefit of overnigh incubation. The probe is added just prior to kinetic measurement (90-120mins), as it measures the change in Oxygen in the sample over time, which is being consumed by the cells/tissue.

The assay must be optimised for the amount of cells / size of tissue per assay volumes, in 150ul if 96-well plate. Therefore, you should prepare samples of different sizes, place tissue samples in wells and measure kinetically to assess different levels of signal increase, thereby identifying the minimal amount of tissue needed for detectable levels of O2 consumption.

Other recommendations:

Standardise tissue weight and Prep. Use normalisation (ATP, total protein, weighing)
Use precision slice cutter
Store tissue samples in carbogen (95% O2), Pre-equilibrate medium
2-4mm x 200µm samples size can be measured in 48WP under >0.2-0.4ml of mineral Oil. (or similar in 24-well plates)
Pre-warm culture media and instruments at 37°C
Use continuous shaking on the fluorescence plate reader.
Use Top reading

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