Incucyte® Mitochondrial Membrane Potential Assay
Mitochondrial membrane potential (MMP or ΔΨm ) is an essential component for maintaining the electrochemical potential of hydrogen ions needed to synthesize ATP via the electron transport chain and has been shown to be an important factor in mitochondrial health. Under normal physiological activity, MMP remains relatively stable. However, long-term disturbance has detrimental effects on cellular health and viability and lead to various pathophysiologies. Monitoring MMP provides a sensitive and early indicator of mitochondrial integrity and is crucial in identifying mitochondrial dysfunction related to pathological diseases such as diabetes, cancer, and neurodegeneration.
Common assays used to evaluate MMP often rely on indirect, concatenated end-point measurements that cannot be easily verified by morphological changes. The Incucyte® Mitochondrial Membrane Potential Assay enables real-time detection of transient and long-term changes in MMP in live cells and can be multiplexed with cell labeling, apoptosis, or cytotoxicity reagents for additional measurements of cell health. Track changes in MMP continuously to unravel the complexities of cellular homeostasis and disease.
Application
Introducing Incucyte® Mitochondrial Membrane Potential Assay
An integrated solution to automatically visualize and quantify changes in MMP in real time, all within your cell culture incubator. Similar to TMRE and TMRM assays, the cell-permeable Incucyte® MMP Orange Reagent accumulates in active mitochondria in proportion to the ΔΨm generated by the electrochemical gradient across the mitochondrial membrane. A drop in MMP denotes a mitochondrial disruption in function and/or integrity and can also be used as an early indicator of apoptosis.
This assay kit utilizes two control compounds: FCCP, an uncoupling agent that transports H+ ions across the mitochondrial membrane and results in depolarization (indicated by a decrease in fluorescence), and Oligomycin A, an inhibitor of ATP synthase which results in hyperpolarization (indicated by an increase in fluorescence).
Key Advantages
- Evaluate mitochondrial stress - Make kinetic measurements of mitochondrial stress with a simple mix-and-read protocol
- Measure changes in MMP in live cells - Generate automated, quantitative, and reproducible MMP measurements in live cells – all within cell culture incubator
- Monitor morphological changes and multiplex with cell health reagents - Visualize morphological changes over time in tandem with apoptosis or cytotoxicity readouts to evaluate alterations in cell health associated with metabolic changes
Evaluate mitochondrial stress
Utilizing the Incucyte® MMP Orange Reagent Kit, rapidly visualize and quantify changes in MMP using a simple mix-and-read protocol.
Measure changes in MMP in live cells
Generate automated, quantitative, and reproducible MMP measurements in live cells. Quantify changes in MMP by measuring fluorescence intensity over time.
Figure 1. Kinetic monitoring of mitochondrial membrane potential. HeLa cells were treated with Vehicle, Camptothecin, FCCP, or Oligomycin in the presence of the Incucyte® MMP Orange Dye to monitor compound effects on mitochondrial membrane potential over time (A). Histograms of mean fluorescence intensity for control and drug treated wells at 0 and 40 hr highlight compound-induced changes in mitochondrial polarization (B). Label-free cell counting was performed using Incucyte® Cell-by-Cell Analysis Software (C). HD phase/fluorescent images acquired at 6 hr (Vehicle, FCCP, Oligomycin) or 40 hr (Camptothecin)(D).
Monitor morphological changes and multiplex with cell health reagents
The Incucyte® MMP Orange Reagent Kit can be multiplexed with Incucyte® Caspase 3/7, Annexin V, or Cytotox Dyes to quantify apoptosis or cytotoxicity in addition to visualizing changes in cell morphology over time.
Figure 2. Multiplex mitochondrial membrane potential measurements with cell health readouts. HeLa cells were treated with the protein kinase inhibitor Staurosporine (SSP) in the presence of the Incucyte® MMP Orange and Cytotox NIR Dyes to evaluate compound effects on mitochondrial membrane potential and cytotoxicity in tandem (A). Concentration-response curves for both MMP and Cytotox NIR assays are shown (24 hr data, B). Representative HD phase + fluorescent images of Control, 0.3 µM SSP, and 3 µM SSP treated wells (24 hr timepoint, C).
Ordering Information
Ordering Information
Product | Qty. | Cat. No. |
|---|---|---|
| Incucyte® MMP Orange Reagent Kit | 1 lkit | |
| Incucyte® Cell-by-Cell Analysis Software Module | 1 module |
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Frequently Asked Questions
Much like the TMRE and TMRM assays, the cell-permeable Incucyte® MMP Orange Reagent accumulates in active mitochondria based on the ΔΨm created by the electrochemical gradient across the mitochondrial membrane. A decrease in MMP indicates mitochondrial dysfunction or damage and can also serve as an early sign of apoptosis.
Dysregulation of mitochondrial function has been linked to diseases such as diabetes, cancer, and neurodegeneration, so being able to evaluate mitochondrial integrity can be crucial in understanding the various pathophysiologies.
Yes, the Incucyte® MMP Orange Kit can be used in conjunction with other cell health reagents, such as Incucyte® Caspase, 3/7, Annexin V, or Cytoox Dye to evaluate apoptosis and cytotoxicity, as well as visualizing changes in cell morphology, over time.
This kit includes the Incucyte® MMP Orange Dye along with two control substances: FCCP, an uncoupling agent that moves H+ ions across the mitochondrial membrane, causing depolarization (shown by reduced fluorescence), and Oligomycin A, an ATP synthase inhibitor that leads to hyperpolarization (reflected by increased fluorescence).
No. Due to different acquisition methods (metabolism using using dual excitation/ single emission), and that both the Incucyte® MMP and Incucyte® CytoATP are read in the orange channel, these two assays cannot be multiplexed.