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MITO-ID® Extracellular O2 Sensor kit

High-throughput plate assay used to detect  mitochondria dysfunction through oxygen consumption measurements in isolated mitochondria
 
ENZ-51044-K100 100 Reactions 320.00 USD
 
ENZ-51044-K500 500 Reactions 1,454.00 USD
Do you need bulk/larger quantities?
 
  • Phosphorescent, oxygen sensitive probe allows for high through-put and real-time oxygen consumption readout
  • Direct real time kinetic analysis of extracellular oxygen consumption rate (OCR) as a measure of mitochondrial function and respiration
  • Mitochondrial Function and Drug Toxicity
  • For use with isolated mitochondria
MITO-ID® Extracellular O2 Sensor Kit contains a phosphorescent oxygen sensitive reagent enabling high-throughput and real-time oxygen consumption readout. In this assay, MITO-ID® Extracellular O2 Sensor Probe 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. Through the process of mitochondrial respiration, oxygen is depleted in the surrounding solution/environment, which is seen as an increase in phosphorescence signal. The addition of oil (supplied) is used to limit back diffusion of ambient oxygen. Rates of oxygen consumption are calculated from the changes in fluorescence signal over time. The reaction is non-destructive and fully reversible (neither the oxygen sensitive reagent nor oxygen are consumed), facilitating measurement of time courses and drug treatments.
ENZ-51044 Fig10
Schematic representation on overall setup of using the MITO-ID® Extracellular O2 Sensor Kit in determining mitochondrial toxicity from isolated mitochondria. (Note: can test different respiration states)
ENZ-51044 Fig5
Figure shows the oxygen consumption and cell growth (OD 600) threshold of E. Coli. Results indicate that seeding concentration is dependent on oxygen consumption and replication rate. This type of data and approach can be useful when analyzing a shift from aerobic to anaerobic metabolism for various microbes.
ENZ-51044 Fig2
Panel A represents monitoring of mitochondria function (from isolated mitochondria) with various compounds. Results demonstrate a decrease in phosphorescence signal when cells are treated with classical inhibitors such as Oligomycin and Antimycin. This suggests a decrease in oxygen consumption or ETC activity. Dose dependent measurements are shown in panel B.
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ENZ-51044 Fig10 ENZ-51044 Fig5 ENZ-51044 Fig2

Product Specification

Applications:Fluorescent detection, HTS
 
Application Notes:For the measurement of extracellular oxygen consumption (OCR) in isolated mitochondria, tissues, enzymes, bacteria and other small microorganisms.
 
Handling:Avoid exposure to light. Reconstituted probe should be stored at -20°C and used within one month.
 
Shipping:Shipped on Blue Ice
 
Short Term Storage:+4°C
 
Long Term Storage:-20°C
 
Kit/Set Contains:MITO-ID® Extracellular O2 Sensor Probe, Vial
MITO-ID® Oil, Bottle
 
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® O2 Extracellular Sensor kit easily overcomes this limitation.
 
Technical Info/Product Notes:It is recommended to purchase Prod. No. ENZ-51045 for cell-based applications. Probe is available as standalone (Prod. No. ENZ-51047).
 
Protocol:Overview
NOTE:Use a plate block heater for plate preparation and pre-warm plate reader to measurement temperature (typically 37°C).

Reagent Preparation
For use with isolated mitochondria, prepare measurement buffer (pH 7.4) according to the following table:

Constituent Concentration (mM)
Sucrose 250
KCl 15
EGTA 1
MgCl2 5
K2HPO4 30
 

NOTE: We recommend that all culture media and stock solutions to be used are pre-warmed to the assay temperature (typically 37°C).

Isolated Mitochondria Sample Preparation
For Isolated Mitochondria, dilute to the desired concentration (typically in the range of 0.125-1.5 mg/ml final concentration, depending on the substrate(s) used and whether measuring Basal [state 2] or ADP-stimulated respiration rate [state 3]) in measurement buffer and add 150 μl to each test well.

Initial Isolated Mitochondria Assay Optimizations
Prepare a six point dilution series of mitochondrial preparation in respiration buffer in 1.5 ml total volume for each concentration.
Typical final substrate concentrations of 25 mM (succinate) or 12.5/12.5 mM (glutamate/malate). Final ADP concentration: 1.65mM

STEP 1: For Isolated Mitochondria: dissolve substrate (succinate or glutamate/malate) and ADP in measurement buffer and add 50 µl to test wells giving a final substrate concentration of 25 mM (succinate) or 12.5/12.5 mM (glutamate/malate) and a final ADP concentration of 1.65 mM. NOTE: We recommend always leaving two wells (H11andH12) free from the addition of MITO-ID® Extracellular O2 Sensor Probe, for use as Blank Controls. Add 200 µl measurement buffer (isolated mitochondria assay) to these Blank Control wells.

STEP 2: Add 10 µl reconstituted MITO-ID® Extracellular O2 Sensor Probe to each well, except those wells for use as Blank Controls. Add 10 µl of fresh culture media to these Blank Control wells.

STEP 3: Test compound stock or vehicle (typically 1-10 µl) may be added at this point if desired. NOTE: We recommend keeping the volume of added compound low to minimize any potential effects of solvent vehicle.

STEP 4: Promptly seal each well by adding two drops (or 100µl) pre-warmed mineral oil, taking care to avoid air bubbles. NOTE: Plate preparation time should be kept to a minimum.

STEP 5: Insert the prepared plate into a fluorescence plate reader pre-set to measurement temperature (typically 37°C).

STEP 6: For Isolated Mitochondria: measure MITO-ID® Extracellular O2 Sensor Kit at 1.5 min intervals for 10-30 minutes. Note: Mitochondria are freshly prepared as per user’s protocol and should not be left on ice longer than recommended in the literature. Measurement buffers should be prepared freshly on the day of measurement.
 

Product Literature References

Enzymatic assays for probing mitochondrial apoptosis: Z. Wang, et al.; Methods Mol. Biol. 1265, 407 (2015), Abstract;
Multivalent display of pendant pro-apoptotic peptides increases cytotoxic activity: D.S. Chu, et al.; J. Control Release 205, 155 (2015), Application(s): Microplate measurements of mitochondria respiration using mitochondria suspension, 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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