Cellular Metabolism, O2 consumption and toxicity
MitoXpress® Xtra – Oxygen Consumption Assay [HS Method]
MitoXpress® Xtra – Oxygen Consumption Assay [HS Method] is a highly flexible 96 or 384-well fluorescence plate reader-based approach, for the direct, real-time analysis of cellular respiration and mitochondrial function. The easy-to-use MitoXpress® Xtra assay allows measurement of extracellular oxygen consumption rates (OCR) with whole cell populations (both adherent and suspension cells), isolated mitochondria, permeabilised cells and a wide range of 3D cultures including: tissues, small organisms, spheroids, scaffolds and matrixes. The assay is also suitable for measurement of isolated enzymes, bacteria, yeasts and moulds.
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In this assay, MitoXpress® Xtra is quenched by O2 through molecular collision, and thus the amount of fluorescence signal is inversely proportional to the amount of extracellular O2 in the sample. Rates of oxygen consumption are calculated from the changes in fluorescence signal over time. The reaction is non destructive and fully reversible (neither MitoXpress® Xtra nor O2 are consumed), facilitating measurement of time courses and drug treatments.
The flexible plate reader format, allows multiparametric or multiplex combination with other products, as well as combining with commonly available reagents to measure glycolysis, LDH, JC-1, MMP (Ѱ), ROS, and cellular ATP. For example, MitoXpress® Xtra in combination with Luxcel’s pH-Xtra® – Glycolysis Assay (Cat No. PH-100) allows the simultaneous real-time measurement of mitochondrial respiration and glycolysis and analysis of the metabolic phenotype of cells and the shift (flux) between the two pathways under pathological states.
MitoXpress® Xtra Application Notes:
pH-Xtra™ Glycolysis Assay
The pH-Xtra™ Glycolysis Assay is an easy to use, highly flexible 96 or 384-well fluorescence plate reader-based approach for the direct, real-time, kinetic analysis of extracellular acidification rates (ECA / ECAR). As lactate production is the main contributor to this acidification, ECA measurements are a convenient and informative measure of cellular glycolytic flux. Such measurements offer an important insight into the central role played by altered glycolytic activity in a wide array of physiological and pathophysiological processes, including cellular adaptation to hypoxia and ischemia, and the development and progression of tumorigensis.
The pH-Xtra™ reagent is chemically stable and inert, water soluble and cell impermeable. It exhibits a positive signal response (increased signal with increased acidification) across the biological range (pH6 – 7.5), which coupled with its spectral and response characteristics, make pH-Xtra™ the ideal choice for flexible, high-throughput assessment of ECA. This performance facilitates sensitive robust microtitre-plate based measurements, thereby overcoming many of the problems associated with the more cumbersome potentiometric pH approach. Rates of extracellular acidification are calculated from changes in fluorescence signal over time and as the measurement is non-destructive and fully reversible (pH-Xtra™ reagent is not consumed), measurement of time courses and multiple drug treatments are possible.
The flexible plate reader format also allows multiparametric or multiplex combinations with other products, and with other commonly available reagents, thereby facilitating parallel kinetic measurements of parameters such as ECA, mitochondria membrane potential (ΔѰm), O2 consumption or ROS generation, followed by the end point measure of parameters such as ATP content or cell membrane integrity, all on the same test cells. For example, the combination of Luxcel’s MitoXpress Xtra® – Oxygen Consumption Assay [HS Method; Cat No. MX-200] and pH-Xtra™ Glycolysis Assay allows the simultaneous real-time measurement of the interplay between mitochondrial respiration and glycolysis. This facilitates the determination of a cell’s metabolic phenotype and the quantification of perturbations in the balance between glycolysis and oxidative phosphorylation under various stimuli or pathological states.
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pH-Xtra™ Application Notes:
MitoXpress® Intra – Intracellular Oxygen Assay
MitoXpress® Intra - Video Protocol
MitoXpress® Intra – Application Notes
Molecular oxygen is the key substrate of aerobic metabolism. Knowledge of cell oxygenation is therefore central to a detailed understanding of the cellular metabolic response to a particular treatment or manipulation.
Intracellular measurements have proven very difficult to date, requiring the use of invasive, laborious, low-throughput, technically challenging techniques which in turn have limited the use of such measurements within biological research.
Now for the first time, molecular O₂ can be conveniently monitored within the cell monolayer on a plate reader in a non-invasive, high-throughput manner and in real-time.
This is achieved using the new MitoXpress®-Intra probe and provides a powerful tool for the detailed investigation to this most critical of biological parameters.
MitoXpress®-Intra is a powerful tool for the monitoring of cell oxygenation, mitochondrial function and the metabolic implications of cell signalling;
having been shown to facilitate the real-time assessment of transient changes in cell respiration, oxygen gradients and physiological responses across a range of cell models.
Specifically, it facilitates the measurement of cellular oxygenation; a critical parameter across many fields of research including hypoxia and cancer metabolism.
- Self penetrating ability with high self-loading efficiency
- Suitable for a range of cell types
- Plate reader compatible allowing the analysis of multiple samples
- Can be used in parallel with the MitoXpress® extracellular probe
- Does not require specialised imaging equipment
- Complements other intracellular parameters such as ROS and mitochondrial membrane potential
MitoXpress® Fatty acid oxidation assay
Fatty acid oxidation (FAO) is the primary metabolic pathway in a variety of tissues, becoming particularly important during periods of glucose deprivation. In organs such as liver and skeletal muscle, FAO can provide over 75% of cellular ATP while in cardiac tissue it can be responsible for up to 90% of cellular energy requirements FAO is also now acknowledged as a key factor in cancer metabolism and is also implicated in drug-induced microsteatosis.
Fig. 1 Fatty acit oxidation in mitochondria
The primary pathway for the degradation of fatty acids is mitochondrial fatty acid β oxidation. Longchain fatty acids (LCFAs) are imported into the mitochondria as acyl carnitine. Once inside, acylCoAs are released to undergo an iterative four-step oxidation until the entire chain is oxidized to acetylCoA, while carnitine returns to facilitate further LCFA transport. The Acetyl CoA produced typically enters the TCA, although in liver it can also fuel the production of ketone bodies, an important energy source for other tissues. Both TCA and β oxidation contribute to the pool of reducing equivalents (NADH and FADH2) which, in turn, drive the activity of the ETC and subsequent ATP generation.
To facilitate the convenient measurement of FAO-driven respiration, AMSBIO offers MitoXpress® Xtra FAO Kit (MXC-500). The kit is designed for use with the MitoXpress® Xtra HS - Oxygen Consumption Assay (MX-200) and contains the 18C unsaturated fatty acid Oleate as substrate, supplied as a 2:1 BSA conjugate. The kit also contains a buffer tablet and L-Carnitine for convenient preparation of measurement media and two FAO modulators, Etomoxir and FCCP. Etomoxir inhibits CPT1 thereby preventing Oleate import (Fig. 1), limiting the supply of reducing equivalents to the ETC, and, in turn, reducing oxygen consumption. The remaining ETC activity is driven by non-long chain FAO. FCCP treatment induces maximal ETC activity by dissipating the mitochondrial membrane potential, while the increased demand for reducing equivalents causes a concomitant increases in FAO activity. This increase is limited where exogenous LCFA is unavailable or where import is inhibited.
Chapple, Sarah J., et al. "Bach1 differentially regulates distinct Nrf2-dependent genes in human venous and coronary artery endothelial cells adapted to physiological oxygen levels." Free Radical Biology and Medicine (2015).
Forman, Henry J., and Kelvin JA Davies. "Commentary on “Bach1 differentially regulates distinct Nrf2-dependent genes in human venous and coronary artery endothelial cells adapted to physiological oxygen levels” by Chapple, et al." Free Radical Biology and Medicine (2016).
Disclaimer:MitoXpress® Xtra and pH-Xtra™ are trademarks of Luxcel Biosciences