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MITO-ID® Extracellular pH Sensor Kit

A pH sensitive phosphorescent probe that can monitor cellular lactic acid extrusion, resulting from glycolysis, as a measure of glycolytic flux  (ECA/ECAR)
 
ENZ-51048 1 Kit 290.00 USD
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  • For use on standard fluorescence plate readers (96 or 384 well format), for high-throughput analysis.
  • Real-time kinetic analysis of extracellular acidification rates (ECA / ECAR)
  • Measures alterations in glucose metabolism and glycolytic function, such as is seen in Warburg.
  • For use with whole cells (adherent and suspension), permeabilized cells, 3D cultures, scaffolds and tissues.
  • Multiplexing with Mito-ID® Extracellular O2 Sensor Kit (High Sensitivity) (Prod. No. ENZ-51045) 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.
The MITO-ID® Extracellular pH Sensor Kit provides an easy “mix and measure” assay which can be used to detect changes in pH (Extracellular acidification) from cell populations using a 96/384 well plate system. The probe phosphorescence signal is modulated by pH such that increased acidifications causes increased phosphorescence signal.

Dual time resolved measurements is used to assess alterations in glucose metabolism, glycolytic inhibition and mitochondrial dysfunction. It has been shown that mitochondrial toxins (electron transport inhibitors) prevent or restrict aerobic ATP generation by the ETC. To compensate for this loss of cellular ATP, cells respond by increasing their glycolysis activity which directly results to an increase in acidification. This indicator can be used as a confirmatory parameter when investigating drug induced mitochondria dysfunction. Metabolic changes can play a critical role in a variety of disease states and toxicities. Insight into the two main cellular ATP generating pathways; glycolysis and oxidative phosphorylation, therefore is particularly informative when examining metabolic perturbations.

Use with MITO-ID® Extracellular O2 Sensor Kit (High Sensitivity) (Prod. No. ENZ-51045) and / or ApoSENSOR™ Cell viability assay kit (Prod. No. ALX-850-247) to simultaneously measure mitochondrial respiration and glycolytic flux, to monitor bioenergetics and metabolism.
ENZ-51048 ECA
Extracellular Acidification rate profiles (ECA) are shown above for A549 cells seeded at 0, 10,000, 20,000, 40,000, 60,000, 80,000 cells / well.
ENZ-51048 Fig2
HepG2 cells were cultured in L15 medium at variable seeding concentrations (1.2x104 to 1x105). Results demonstrate that as cell concentrations increase, the probe phosphorescence levels decrease which is indicated by the lifetime measurements of the probe. As ETC function decreases, glycolysis levels is expected to increase to compensate for the loss of ATP.
ENZ-51048 Fig3
Acidification profiles of HepG2 cells treated with glucose transport inhibitors (2DG, Oxamate) or mitochondrial inhibitor (Antimycin) relative to control. Results show a decrease in acidification when cells are treated with glycolysis inhibitors.
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-51048 glycolysis
MITO-ID® Extracellular pH Sensor Probe paired with the MITO-ID® Extracellular O2 Sensor Kit (ENZ-51045) 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-51048 workflow diagram
Workflow Diagram of MITO-ID® Extracellular pH Sensor Probe
ENZ-51048 ex em
Excitation and Emission spectra of MITO-ID® Extracellular pH Sensor Probe. Left panel shows normalized excitation (Ex 340-410nm; Peak 360-380nm). Right panel show emission maxima (Em 590, 615 and 690nm) fold increase between pH6.0 and pH7.5)."
Please mouse over
ENZ-51048 ECA ENZ-51048 Fig2 ENZ-51048 Fig3 ENZ-51044 Fig9 ENZ-51048 glycolysis ENZ-51048 workflow diagram ENZ-51048 ex em

Product Specification

Applications:Fluorescent detection, HTS
 
Application Notes:For the measurement of extracellular acidification rate (ECA/ECAR) in whole cells (adherent and suspension), permeabilized cells, 3D cultures, scaffolds and tissues.
 
Handling:Avoid exposure to light. Store MITO-ID® Extracellular pH Sensor Probe between +2°C to +8°C.
Reconstituted product can be aliquoted in water and stored at -20°C. Use within one month (avoid freeze/thaw cycles).
 
Shipping:Shipped on Blue Ice
 
Short Term Storage:+4°C
 
Long Term Storage:+4°C
 
Kit/Set Contains:MITO-ID® Extracellular Sensor Probe (solid form)
Respiration Buffer Tablet (1 tablet)
 
Scientific Background:As lactate production is the main contributor to acidification, extracellular 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 pathological processes, including cellular adaptation to hypoxia and ischemia, and the development and progression of tumorigenesis.
 
Technical Info/Product Notes:Application Notes:
Measuring Glycolysis as Extracellular Acidification

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.
  1. For adherent cells, seed cells in a 96 well plate at a density (typically 30,000-80,000 cells / well) in 200µl culture medium. For new cell types, we recommend setting up a titration to select the optimum cell seeding density.
    For suspension cells, seed on the day of assay in 150µl respiration buffer at a density of approx. 250,000-500,000 cells / well.
    Note: Where cells are cultured in a CO2 incubator overnight, it is important to purge the media and plastic ware of CO2 prior to conducting the MITO-ID® Extracellular pH Sensor Probe as residual CO2 may contribute to acidification. Perform a CO2 purge, by incubating cells in a CO2 free incubator at 37°C with 95% humidity, approx. 3 hours prior to performing the measurement.
  2. Reconstitute transparent contents of the MITO-ID® Extracellular pH Sensor Probe in 1ml of Respiration Buffer (1mM K-phosphate, 20mM Glucose, 70mM NaCl, 50mM KCl, 0.8mM MgSO4, 2.4mM CaCl2), gently aspirating 3-4 times.
  3. Prepare test compounds, controls and dilutions as desired. Typical controls are oxamic acid (inhibitor; decrease ECA), FCCP (ETC uncoupler; increases ECA) and glucose oxidase (GOx; signal control).
    Note: We recommend that all culture media and stock solutions to be used in the assay are pre-warmed at 37°C prior to use. Use a plate block heater for plate preparation and pre-warm the fluorescence plate reader to measurement temperature.
  4. Remove spent culture medium from all assay wells and wash cells twice, using 100µl of Respiration Buffer per well for each wash. After removing the second wash, replace with 150µl of fresh Respiration Buffer.
    Note: We recommend always leaving two well (H11 and H12) free from the addition of MITO-ID® Extracellular pH Sensor Probe, for use as Blank Controls. Add 150µl of Respiration Buffer to these Blank Control wells also.
  5. Add 10µl reconstituted MITO-ID® Extracellular pH Sensor Probe to each well, except those wells for use as Blank Controls. Add 10µl of Respiration Buffer to these Blank Control wells.
  6. Test compound stock or vehicle (typically 1-10µl) may be added at this point if desired.
    Note: We recommend keeping the volume of added compound low to minimise any potential effects of solvent vehicle.
  7. Read the plate immediately in a fluorescence plate reader. The plate should be measured kinetically for >120 minutes. When the measurement is completed, remove the plate and save measured data to file.
 

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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