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MITO-ID® Extracellular O2 Sensor Kit (High Sensitivity)

High-throughput cell-based  assay used to detect mitochondria dysfunction through real-time oxygen consumption measurements in whole cells
 
ENZ-51045-K100 100 Reactions 320.00 USD
 
ENZ-51045-K500 500 Reactions 1,454.00 USD
Do you need bulk/larger quantities?
 
  • For use on standard fluorescence plate readers (96 or 384 well format), for high-throughput analysis.
  • Direct real-time kinetic analysis of extracellular oxygen consumption rates (OCR) as a measure of cellular respiration and mitochondrial function.
  • Cellular Activity (Mitochondrial Function), Metabolism, Warburg, Apoptosis.
  • For use with whole cells (adherent and suspension), isolated mitochondria, permeabilized cells, 3D cultures, scaffolds and matrixes, tissues, small organisms, isolated enzymes, bacteria, yeasts and molds.
  • Multiplexing with Mito-ID® Extracellular pH Sensor Probe allows the simultaneous real-time measurement of mitochondrial respiration and glycolysis, and the analysis of the metabolic phenotype of cells, and the shift (flux) between the two pathways under pathological states.
  • Combine with Mito-ID® Membrane potential cytotoxicity kit to confirm mitochondrial specific toxicity
MITO-ID® Extracellular O2 Sensor Kit (High Sensitivity), contains a phosphorescent oxygen sensitive reagent enabling high-throughput and real-time oxygen consumption readout. In this assay, MITO-ID® Extracellular O2 SensorProbe is quenched by oxygen, through molecular collision, and thus the amount of fluorescence signal is inversely proportional to the amount of extracellular oxygen 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 MITO-ID® Extracellular O2 SensorProbe nor oxygen are consumed), facilitating measurement of time courses and drug treatments. The addition of high-sensitivity mineral oil (supplied) is used to limit back diffusion of ambient oxygen. The flexible plate reader format, allows multiparametric or multiplex combination with a range of other reagents.
  • Use with MITO-ID® Extracellular pH Sensor Probe (Prod. No. ENZ-51048) and / or ATP Assay Kit (Prod. No. ALX-850-247) to simultaneously measure mitochondrial respiration and glycolytic flux, to monitor bioenergetics and metabolism.
  • Use with MITO-ID® Membrane potential cytotoxicity kit (Prod. No. ENZ-51019) to simultaneously measure mitochondrial respiration and mitochondrial membrane potential (apoptosis)
  • For the intracellular analysis of molecular oxygen concentration and hypoxia, use MITO-ID® Intracellular O2 Sensor Probe (Prod. No. ENZ-51046).
ENZ-51045 dose response
Dose response curve as an example of the data typically produced with this assay. Drug concentration (µM) versus calculated slope (µs/hour) demonstrates that this drug causes inhibitory response on cellular respiration.
ENZ-51044 Fig8
Schematic representation of a possible metabolic response to drug treatment by cells. If mitochondrial toxicity is the primary site of toxic insult, the addition of an inhibitor should show a decrease in oxygen consumption (ETC activity) with an increase in acidification. An increase in acidification is expected since glycolysis will help compensate the decrease in ATP production by the ETC. An uncoupler should show an increase in oxygen consumption with an decrease in acidification. Contrary results would indicate toxicity via multiple mechanisms (possibly by glucose uptake inhibition).
ENZ-51044 Fig11
Assessment of mitochondrial function with MITO-ID® Extracellular O2 Sensor Kit (HS) (A) or traditional ATP assay (B) following treatment with mitochondrial inhibitors (Oligomycin, Rotenone, Antimycin) and uncoupling agent (FCCP). Results illustrate that drug-induced mitochondrial dysfunction is evident immediately post-treatment (A) despite varying levels of viability @ 24 hours by ATP assay (B)
ENZ-51044 Fig9
Cell based assay using MITO-ID® Extracellular O2 Sensor Kit (High Sensitivity) and MITO-ID® Extracellular pH Sensor Probe. A consequence of Mitochondrial toxicity includes, decrease in oxygen consumption (ETC levels) and increase in glycolytic flux (extracellular acidification) to compensate for the decrease in ATP production. While compound 1 has no effect on toxicity, compound 2 shows a dose dependent effect on oxygen consumption (decrease) with an increase in acidification. This represents True" mitochondrial toxicity. The results of compound 3 indicate that the mitochondrial toxicity is not the primary site of toxic insult.
ENZ-51045 glycolysis
MITO-ID® Extracellular O2 Sensor Kit paired with the MITO-ID® pH Sensor (ENZ-51048) allows the simultaneous real-time measurement of mitochondrial respiration and glycolysis and analysis of the metabolic phenotype of cells and flux between the two pathways under pathological states.
ENZ-51045 workflow
Workflow of Mito-ID® Extracellular O2 Sensor Kit (High Sensitivity)
ENZ-51045 ex em
Excitation and Emission spectra of oxygen-sensitive reagent Mito-ID® Extracellular O2 Sensor Probe. Left panel shows normalized excitation (EX 360 – 400nm; Peak 380nm). Right panel shows emission (Em 630 – 680nm; Peak 650nm) in oxygenated and deoxygenated conditions.
ENZ-51045 lifetime profile
Typical Lifetime profiles of MITO-ID® Extracellular O2 Sensor Kit (High Sensitivity) 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. NOTE: If using FCCP, it is recommended to perform a dose titration since FCCP exhibits a bell shaped response"
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ENZ-51045 dose response ENZ-51044 Fig8 ENZ-51044 Fig11 ENZ-51044 Fig9 ENZ-51045 glycolysis ENZ-51045 workflow ENZ-51045 ex em ENZ-51045 lifetime profile

Product Specification

Applications:Fluorescent detection, HTS
Cell-based assays
 
Application Notes:For the measurement of extracellular oxygen consumption (OCR) in whole cells (adherent and suspension), isolated mitochondria, permeabilized cells, 3D cultures, scaffolds and matrixes, tissues, small organisms, isolated enzymes, bacteria, yeasts and molds.
 
Handling:Avoid exposure to light. Once reconstituted, the MITO-ID® Extracellular O2 Sensor Probe should be stored at -20°C. Avoid freeze/thaw cycles. MITO-ID® (High Sensitivity) Oil may be stored at room temperature. Protect from light.
 
Shipping:Shipped on Blue Ice
 
Short Term Storage:+4°C
 
Long Term Storage:+4°C
 
Kit/Set Contains:MITO-ID® Extracellular O2 Sensor Probe
MITO-ID® (High Sensitivity) Oil
 
Scientific Background:The mitochondria plays a central role in cellular metabolism, bioenergetics, and apoptosis. Mitochondrial dysfunction is implicated in numerous disease states and is also a major mechanism of drug-induced toxicity. Oxygen consumption is one of the most informative and direct measures of mitochondrial function. Limitations with the traditional methods of measuring oxygen consumption, includes low throughput. The MITO-ID® Extracellular O2 Sensor Kit (High Sensitivity) easily overcomes this limitation.
 
Technical Info/Product Notes:Application Notes:
Hepatotoxicity: Measuring drug-induced mitochondrial toxicity in HepaRG™ cells using the MITO-ID® Extracellular O2 Sensor Kit

Cardiotoxicity: Assessing Mitochondrial Toxicity in Stem Cell-Derived Cor.4U® Cardiomyocytes

Cellular Energy Flux in Real Time: Optimization of a multi-mode detection model for measuring real-time cellular respiration and mitochondrial function using fluorophoric biosensors
 
Protocol:Overview
NOTE:Use a plate block heater for plate preparation and pre-warm plate reader to measurement temperature (typically 37°C).
STEP 1: For adherent cells, seed cells in a 96 well plate at a density (typically 40,000 – 80,000 cells / well) in 200µl culture medium. Incubate overnight in a CO2 incubator at 37°C.
For suspension cells, seed on the day of assay in 150µl culture medium at a density of ~4 x 106/ml.
STEP 2: Reconstitute contents of the MITO-ID® Extracellular O2 Sensor Probe in 1ml of water, PBS or culture media, gently aspirating 3-4 times.
STEP 3: Remove spent culture medium from all assay wells and replace with 150µl of fresh culture media. We recommend to leave two wells (H11 and H12) free from the addition of the MITO-ID® Extracellular O2 Sensor Probe, for use as Blank Control wells. Add 150µl of fresh culture media to these Blank Control wells.
STEP 4: Add 10µl reconstituted MITO-ID® Extracellular O2 Sensor Probe to each well except Blank Control wells (H11 and H12). Add 10µl of fresh culture media to these Blank Control wells.
STEP 5: Test compound stock may be added at this point if desired.
STEP 6: Promptly seal each well by adding two drops (or 100µl) pre-warmed High sensitivity mineral oil, taking care to avoid air bubbles.
STEP 7: Read plate immediately in a fluorescence plate reader. The plate should be measured kinetically for >90. When the measurement is completed, remove the plate and save measured data to file.
 

Product Literature References

The mitochondrial unfoldase-peptidase complex ClpXP controls bioenergetics stress and metastasis: J.H. Seo, et al.; PLoS Biol. 14, e1002507 (2016), Application(s): Oxygen consumption in various prostate cancer cells, Abstract; Full Text
Transfer of mitochondria from astrocytes to neurons after stroke: K. Hayakawa, et al.; Nature 531, 551 (2016), Application(s): Oxygen consumption in astrocytic particles, Abstract; Full Text
Transgenic expression of the mitochondrial chaperone TNFR-associated protein 1 (TRAP1) accelerates prostate cancer development: S. Lisanti, et al.; J. Biol. Chem. 291, 25247 (2016), Abstract;
Survivin promotes oxidative phosphorylation, subcellular mitochondrial repositioning, and tumor cell invasion: D.B. Rivadeneira, et al.; Sci. Signal. 8, ra80 (2015), Application(s): Oxygen consumption in various tumor cell types, Abstract; Full Text
Deletion of the Mitochondrial Chaperone TRAP-1 Uncovers Global Reprogramming of Metabolic Networks: S. Lisanti, et al.; Cell Rep. 8, 671 (2014), Application(s): Measuring oxygen consumption in primary hepatocytes, Abstract;

General Literature References

High throughput fluorescence assays for the measurement of mitochondrial activity in intact human neuroblastoma cells: A.J. Woollacott & P.B. Simpson; J. Biomol. Screen 6, 413 (2001), Abstract;
A New Method for the Cytofluorometric Analysis of Mitochondrial Membrane Potential Using the J-Aggregate Forming Lipophilic Cation 5,5′,6,6′-Tetrachloro-1,1′,3,3′-tetraethylbenzimidazolcarbocyanine Iodide (JC-1): A. Cossarizza, et al.; Biochem. Biophys. Res. Commun. 197, 40 (1993), Abstract;
Intracellular heterogeneity in mitochondrial membrane potentials revealed by a J-aggregate forming lipophilic cation JC-1: S.T. Smiley, et al.; PNAS 88, 3671 (1991), Full Text

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