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MITO-ID® Intracellular O2 Sensor Probe

Sensitive fluorescence assay for direct measurement of cellular oxygen concentration
 
ENZ-51046 1 Vial 407.00 USD
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  • Easy, mix and measure, fluorescence (TR-F) microplate reader based assay
  • Measures available oxygen concentration (cellular oxygenation), in the close proximity to cells in culture
  • Cell-penetrating sensor probe, only measures intracellular and pericellular oxygen levels.
  • Applications include Cellular Environment, Hypoxia, Oxidative Stress, Metabolism and Signal Transduction.
  • Use with CYTO-ID® Hypoxia / Oxidative Stress Detection Kit (Prod. No. ENZ-51042)
  • Multiplex with MITO-ID® Extracellular pH Sensor Probe (Prod. No. ENZ-51048)
The MITO-ID® Intracellular O2 Sensor Probe is an oxygen-sensitive nanoparticle chemistry developed for the real-time measurement and monitoring of intracellular and pericellular oxygen concentration (cellular oxygenation), using time-resolved fluorescence (TR-F). The probe is taken up via non-specific energy dependent endocytosis (3-18 hours) and, after washing, the cells are monitored on a TR-F microplate reader.

Probe signal is quenched by molecular oxygen in a non-chemical, reversible manner (probe signal increases with decreased oxygenation and signal deceases with increased oxygenation). Probe signal can be converted to absolute oxygen concentrations or percentage oxygen. The flexible 96 or 384 well microplate reader assay format, allows multiparametric or multiplex combination with a range of other reagents.

Product Specification

Absorbance maximum:360nm-400nm, Peak 380nm
 
Emission maximum:630nm-670nm, Peak 650nm
 
Appearance:Dry solid
 
Solubility:Soluble in sterile water
 
Applications:Fluorescent detection, HTS
 
Application Notes:Cellular Environment, Hypoxia, Oxidative Stress, Metabolism and Signal Transduction
 
Shipping:Shipped on Blue Ice
 
Long Term Storage:+4°C
 
Use/Stability:When reconstituted in sterile water, probe can be stored at +4°C for up to 3 weeks.
 
Handling:Avoid exposure to light. Do not freeze.
 
Scientific Background:Available, cytosolic oxygen concentration significantly influences signal transduction and cellular metabolism – but, majority of in vitro studies at ambient oxygen ignore the oxygen gradient between the atmosphere and medium and between medium and the cell.

The MITO-ID® Intracellular O2 Sensor Probe provides a unique tool to quantitatively monitor the oxygen concentration that cells in culture are actually experiencing. Additionally, where the experimental objective is to monitor cell physiology under defined O2 conditions, MITO-ID® Intracellular O2 Sensor Probe provides the ideal tool to identify the appropriate environmental O2 to achieve this desired cellular O2 concentration as it accounts for the significant impact cell respiration can have on intracellular O2 concentration. 

In combination with plate reader oxygen control units and hypoxia workstations, MITO-ID® Intracellular O2 Sensor Probe allows the possibility to measure, monitor and control oxygenation to better mimic the physiological in vivo environment in 2D and 3D models.
 
Technical Info/Product Notes:The level of available oxygen in the cell significantly influences cell physiology, signal transduction and cellular response to drug treatment. In spite of this knowledge, the majority of in vitro studies culture and study cells at ambient oxygen – ignoring the oxygen gradient between the atmosphere and the medium and between the medium and the intracellular cell environment. As the real oxygen concentration experienced by cells in culture is a function of environmental oxygen concentration, cell metabolism and seeding density, MITO-ID® Intracellular O2 Sensor Probe provides the ideal tool to intelligently modulate these parameters to achieve a desired and specific cellular oxygenation. Similarly, MITO-ID® Intracellular O2 Sensor Probe is an ideal tool to monitor real-time changes in cellular oxygenation in response to treatments that perturb mitochondrial function and cell metabolism.

Application Notes:
Real-time Measurement of Intracellular O2 in Mammalian Cells (BMG LabTech)

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 (BioTek)
 
ENZ-51046 Fig5
HCT116 cells were treated with the agonist, Ryaniodine. Results indicate that with the MITO-ID® (R) Intracellular O2 Sensor probe, changes in intracellular oxygen levels (or ETC) can be detected by lifetime measuremnts. The agonist will cause an increase in ETC activity, therefore increasing lifetime of the probe, which suggests a decrease in intracellular oxygen levels.
ENZ-51046 Fig3
Lifetime measurement values obtained from the MITO-ID® Intracellular O2 Sensor Probe while changing the atmospheric control unit at known oxygen levels. By calibrating the probe, known O2 levels can be correlated with oxygen levels for various applications.
ENZ-51046 workflow diagram
Workflow of MITO-ID® Intracellular MITO-ID® Intracellular O2 Sensor Probe
ENZ-51046 Antimycin FCCP
The effect of Antimycin and FCCP treatment on the oxygen levels at a HEK293T cell monolayer. Antimycin is an ETC inhibitor while FCCP uncouples oxygen consumption from ATP production. Initial probe signal reflects the steady state oxygen level lower than ambient oxygen due to cellular oxygen consumption. Antimycin treatment inhibits this consumption resulting in oxygen diffusing into the monolayer returning it to ambient concentrations. FCCP treatment causes an increase in oxygen consumption resulting in in a rapid decrease in the concentration of oxygen at the cell monolayer until a new steady state is reached
ENZ-51046 O2 level
Monitoring changes in monolayer oxygenation at decreasing oxygen concentration. Oxygen concentration is stepped between 21% and 1%, with an atmospheric control unit. The MITO-ID® Intracellular O2 Sensor Probe signal reflects the actual oxygen concentrations at the cell monolayer. Note that cellular respiration drives the monolayer to much lower oxygen concentrations than those imposed.
Note: MITO-ID® Intracellular O2 Probe is simply added ~16 hours prior to drug treatment. The probe is stable and reversible, allowing real-time monitoring of (multiple) drug effects under controlled oxygen conditions.
ENZ-51046 ex em
Excitation spectra for MITO-ID® Intracellular Sensor Probe excitation spectra (left) and emission spectra at 21% and 0% O2 (right).
Please mouse over
ENZ-51046 Fig5 ENZ-51046 Fig3 ENZ-51046 workflow diagram ENZ-51046 Antimycin FCCP ENZ-51046 O2 level ENZ-51046 ex em

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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