The probe is a cationic carbocyanine dye with a polychromatic fluorescent emission in which the dye fluoresces green or red, depending upon mmp. Membrane potential also transmembrane potential or membrane voltage is the difference in electric potential between the interior and the exterior of a biological cell. H9c2 cells were plated at a density of 2 x 104 and cultured overnight. Mitochondrial membrane potential measurement tmrmpmpi this pipeline performs all image processing tasks required for calibration of a tmrm pmpi flipr plasma membrane potential assay kit to millivolts. In recent years, fluorescent dyes for measuring the mitochondrial membrane potential m have become commonly used tools for monitoring changes in this important physiologic mitochondrial parameter as it relates to cells capacity to generate atp by oxidative phosphorylation. A representative fluorescence image of cortical neurons loaded with tmrm. Mitochondrial membrane potential by object spot counting. Structure of cell membrane, resting membrane potential textbooks and resources medical iophysics damjanovich s, fidy.
Mitochondrial membrane potential and reactive oxygen. After staining a cell population with alexa fluor 488 annexin v and mitotracker red cmxros dye in the provided bindin. A homogenous cellbased mmp assay was optimized and performed in a 1536well plate format to screen several compound libraries for mitochondrial toxicity by evaluating the effects of chemical compounds on mmp. If youre behind a web filter, please make sure that the domains. The potential is required for all protein import into the matrix1416, and its elecprotein unfolding by the mitochondrial membrane potential shihai huang, kevin s. Resting membrane potential rmp the human organism is composed of multiple cells, all of them with different components and therefore with differents resting membrane potentialssome of these cells are excitable e. The mitochondrial membrane potential is central to the organelles many functions.
Lack of oxidative phosphorylation and low mitochondrial. Most graphs will start at resting potential 70 mv 5. Mitochondrial respiration and membrane potential are. The slowresponse potentialsensitive probe, dibac 4 3 enters depolarized cells where it binds to intracellular proteins or membrane and exhibits enhanced fluorescence and a red spectral shift. Since, by convention, the potential outside the cell is defined as zero, the resting potential vr is equal to vin. To date, major research effort has been aimed at identifying and. Jc1 staining pattern indicates loss of membrane potential in h9c2 cells treated with fccp. Mitochondrial membrane potential and aging wiley online library. We offer a range of molecular probes products specifically designed to assay mitochondrial membrane potential.
The calculated nernstian equilibrium potential for k,na, and cl establish the boundary conditions for the electrical pd across the cell membrane. Mitochondria with intact membrane potential higher than 100 mv concentrate jc1 into aggregates that fluoresce red, whereas deenergized mitochondria cannot concentrate jc1 and fluoresce green. Measurement of mitochondrial membrane potential using fluorescent rhodamine derivatives russell c. This process is controlled by the membrane potential of this organelle. The next day, cells were stained with jc1 according to the protocol on page 7 and imaged with bioteks cytation 5 cell imaging multi. The kit includes the cationic dye tmre tetramethylrhodamine ethyl ester perchlorate and a mitochondrial membrane potential disruptor cccp carbonyl cyanide 3chlorophenylhydrazone. Alterations in mitochondrial membrane potential during. Mitochondrial membrane potential is important for many mitochondrial processes and is linked to cell health.
Assessment of mitochondrial membrane potential and ros levels in live rat cortical neurons. Toxicity and loss of mitochondrial membrane potential. Their directionality is governed by the mitochondrial membrane potential. If youre seeing this message, it means were having trouble loading external resources on our website. We have presented a stepbystep procedure describing how to determine m and ros in rat cortical neurons using the fluorescent indicators tmrm and h 2 dcfda, respectively. Mitochondrial membrane potential and aging nicholls. After scanning the baseline tmrm fluorescence, neurons were treated with the protonophore fccp 1. In the rest state, the inside of the nerve cell membrane is negative with respect to the outside typically about. Targeting this unique cell population would provide a less toxic approach than regular chemotherapeutic agents that kill bulk rapid proliferating tumor cells and also normal cells which divide rapidly. Utilzing flourometric probes to assess is a common initial approach in determining cell health and may be used to help understand toxicity mechanisms in. Grotyohann department of cellular and molecular physiology, college of medicine, the pennsylvania state university, hershey, pennsylvania 17033 usa. This difference in electrical charge between the inside and outside of the membrane is the basis for many types of. Heterogeneity of m within a single cell may be a sign of a pathology.
Detection of the mitochondrial membrane potential by the. Combining mitochondria targeted probes, click chemistry. How the resting membrane potential is established in a neuron. Mitochondria membrane potential kit jc10 assay for microplate readers catalog number mak159 storage temperature 20 c technical bulletin product description mitochondria generate a potential across their membranes due to the activities of enzymesof the electron transport chain. Pdf the mitochondrial membrane potential or protonmotive force is the central bioenergetic parameter that controls respiratory rate, atp. Why cancer cells have a more hyperpolarised mitochondrial. An ionophore that dissipates the mitochondrial membrane potential of. Learn vocabulary, terms, and more with flashcards, games, and other study tools. The resting membrane potential rmp is due to changes in membrane permeability for potassium, sodium, calcium, and chloride, which results from the movement of these ions across it. Pdf mitochondrial membrane potential and aging researchgate. With respect to the exterior of the cell, typical values of membrane potential, normally given in units of milli volts and denoted as mv, ranges from 40 mv to 80 mv. Mitochondrial membrane potential is a key regulator of cell health.
The electrical nature of the impulse was first suspected by luigi galvani in 1780, when he caused the leg of a frog to contract after stimulating it with an electric charge from the newly developed leyden jar. The presence of diabetes was the only variable significantly associated with mitochondrial membrane potential r0. Respiration of mitochondria is regulated by both, the membrane potential m, as well as by atp which directly inhibits cco in a mindependent way, as shown in fig. Opa1 proteolytic cleavage is regulated by the mitochondrial membrane potential, apoptosis, atp level and mtdna stability. Cell membrane potentials cell membranes in general, and membranes of nerve cells in particular, maintain a small voltage or potential across the membrane in its normal or resting state. Membrane potential indicators thermo fisher scientific us. Pdf the mitochondrial membrane potential m generated by proton pumps complexes i, iii and iv is an essential component in the.
When current injection stim is present, move the membrane potential upward to firing threshold. Sep 30, 2014 cancer stem cells cscs are believed as the initiators of the occurrence, development and recurrence of malignant tumors. Structure of cell membrane, resting membrane potential textbooks and resources medical iophysics damjanovich s, fidy j, szollosi j editors medicina press, 2009. The respiration of rat heart mitochondria with glutamate plus malate is stimulated by adp state 3 respiration and inhibited after conversion of adp into atp state 4 respiration. Mitochondrial membrane potential indicators to detect apoptosis. Depolarization below a certain m may indicate impaired mitochondrial function and is a prerequisite for mitophagy 9, 14. Mitochondria membrane potential kit mak159 technical. Protein unfolding by the mitochondrial membrane potential.
Butachlor induced dissipation of mitochondrial membrane. For the time during which membrane conductance to a particular ion increases, move the membrane potential toward the nernst potential for. With respect to the exterior of the cell, typical values of membrane potential, normally given in units of milli volts and denoted as mv, ranges from 40 mv to 80 mv all animal cells are surrounded by a membrane composed. Conversely, hyperpolarization is indicated by a decrease in fluorescence. Pdf relation between mitochondrial membrane potential. As such, the m is a key indicator of cell health or injury. The next day, cells were stained with jc1 according to the protocol in section of fluorescence. Assessing the mitochondrial membrane potential in cells and in. Jan 01, 2004 mitochondrial membrane potential is a key indicator of cellular viability, as it reflects the pumping of hydrogen ions across the inner membrane during the process of electron transport and oxidative phosphorylation, the driving force behind atp production. The acrosome reaction, on the other hand, was always associated with sperm plasma membrane potential depolarization, whether it was induced. For other cell types, it is important to empirically determine the final concentration and loading time for tmrm or h 2 dcfda. Both a receptor potential and an action potential change the membrane potential. Study of mitochondrial membrane potential, reactive oxygen.
Membrane potential resting membrane potential article. Mitochondrial membrane potential and reactive oxygen species. Measurement of plasma membrane and mitochondrial potentials in. The resting membrane potential barbados underground. They were treated with fccp at the concentrations indicated and stained with tmre at 25 nm a, no fccp and b, 10 m fccp or 100 nm c and hoechst dye item no. Ion channels are involved in the control of membrane potential. This study examined changes in mitochondrial membrane potential m throughout the stages of preimplantation development in mouse embryos conceived either in vivo or in vitro and human embryos donated to research from ivf. Mcf7 cells causes them to resemble cv1 cells by decreasing uptake. The presence of diabetes was the only variable significantly associated with. The resting membrane potential of a cell is the electrochemical state of a cell neuron at rest, i. The membrane potential of a cell at rest is called the resting membrane potential. Cells were grown in 25cm 2 dishes until 90% confluence and either kept in the medium alone control or treated with 0.
H9c2 cells were plated and allowed to grow to about 70% confluence. The next day, cells were stained with jc1 according to the protocol in section of fluorescence microscopy and imaged with bioteks cytationtm 5. Relation between mitochondrial membrane potential and ros formation. Electrotonic and action potentials video khan academy. Do all mitochondria within one cell necessarily adopt the same membranepotentials. Mitochondria are cellular organelles regulating metabolism and cell death pathways. Because of its physical location, the membrane potential across the inner mitochondrial membrane would be able to act on precursors before they reach mthsp70. Mitochondrial dysfunction and oxidative stress occur early in all major neurodegenerative diseases, and there is strong evidence that this dysfunction has a causal role in disease pathogenesis. The mitochondrial membrane potential or protonmotive force is the central bioenergetic parameter that controls respiratory rate, atp synthesis. Generation of resting membrane potential stephen h.
The magnitude of the potential was assessed by monitoring fluorescence of the dye jc1. Mitochondria are found in every cell of the human body except red blood cells, and convert the energy of food molecules into the atp that powers most cell functions mitochondrial diseases take on unique characteristics both because of the way the diseases. Once the membrane is polarized, it acquires a voltage, which is the difference of potentials between intra and extracellular spaces. Mitochondrial membrane potential and aging nicholls 2003. Mitochondrial and plasma membrane potentials cause unusual. Cancer stem cells cscs are believed as the initiators of the occurrence, development and recurrence of malignant tumors. Determination of mitochondrial membrane potential and. Intracellular heterogeneity in mitochondrial membrane potentials. In addition, posthoc immunocytochemistry images showing the same view field after immunofluorescence. This is a pdf file of an unedited manuscript that has been accepted for publication. So if you had a voltmeter measuring the potential difference across the membrane, so if you took this voltage minus this voltage right over here, the voltage between this and that, you would get negative lets say for the sake of argument, lets say it would measure, it would average about negative 70 millivolts.
Table 4 shows the results of our univariate analyses for predictors of mitochondrial membrane potential and mass. Toxicity and loss of mitochondrial membrane potential induced. Mitochondrial membrane potential m is the driving force for mitochondrial atp synthesis. Mitochondrial membrane potential, analytical biochemistry 2017, doi. This flow cytometry product provides a rapid and convenient assay for two hallmarks of apoptosis phosphatidylserine externalization and changes in mitochondrial membrane potential. Fccp is a potent mitochondrial oxidative phosphorylation uncoupler and serves as positive control for depolarization of mitochondrial membrane or for apoptosis.
Perturbing mmp can lead to mitochondrial dysfunction and disrupt atp production, cellular homeostasis, and induce cell death. We used a watersoluble mitochondrial membrane potential indicator mmpi to detect changes in mmp in hepg2 cells. The mitochondrial membrane potential assay kit ii is a fluorescent assay that detects the mitochondrial membrane potential in living cells. Predictors of mitochondrial membrane potential and mitochondrial mass.
Assessment of mitochondrial membrane potential in yeast. Mitochondrial membrane potential probes and the proton. The mitochondrial membrane potential m generated by proton pumps complexes i, iii and iv is an essential component in the process of energy storage during oxidative phosphorylation. In the presence of tariquidar, measurement of the jc1 fluorescence revealed similar levels of mitochondrial membrane potential in pgp. Considering that the loss of mitochondrial membrane potential is a typical characteristic of apoptotic cells, it was used to elucidate a possible mode of parasite death. Decreased mitochondrial membrane poten tial m has been found in a variety of aging cell types from several mammalian species. Altered mitochondrial membrane potential, mass, and. This study describes the optimization and validation of a lowconcentration rh123 staining protocol for the flowcytometric assessment of mitochondrial membrane potential m changes in whole.
Mitochondrial membrane potential identifies cells with. Membrane potentials definition of membrane potentials by. Which are set by the concentrations of the participating reactants, as shown in equations 111 4, 50. Measurement of mitochondrial membrane potential using.
It is the transmembrane potential of the resting cellat which there is no net movement of a particular ion. Increased depolarization results in additional influx of the anionic dye and an increase in fluorescence. A distinctive feature of the early stages of apoptosis is the disruption of the mitochondria, including changes in membrane and redox potential. Mitochondrial membrane potential in aging cells karger publishers. Neuron depolarization, hyperpolarization, and action potentials. In living cells, jc1 dye crosses the plasmatic membrane as monomers, penetrating into the mitochondria. Mitochondria membrane potential kit mak159 technical bulletin. Indeed, opening of the mitochondrial permeability transition pore has been demonstrated to induce depolarization of the transmembrane potential m, release. The total force driving protons into the mitochondria i. Mitochondrial diseases are a group of disorders caused by dysfunctional mitochondria, the organelles that generate energy for the cell. In pacemaker cells this is a point of hyperpolarization. It is the transmembrane potential of the resting cellat. Mitochondrial membrane potential apoptosis kit, with. The mitochondrial membrane potential or protonmotive force is the central bioenergetic parameter that controls respiratory rate, atp synthesis and the generation of reactive oxygen species, and is itself controlled by electron transport and proton leaks.
Mitochondrial fission involves another gtpase called drp1. Wright department of physiology, college of medicine, university of arizona, tucson, arizona 85724 received 2 july 2004. All cells exhibit an electrical potential across their membranes called a membrane potential mp. Membrane potential and action potential every cell in the body is electrically active. Mitochondrial membrane potential can be quantified by imaging. The means by which nerves conduct impulses have been puzzling researchers for centuries.
Mar, 2012 membrane potential and action potential every cell in the body is electrically active. Assessment of mitochondrial membrane potential mitochondrial membrane potentials m were measured by means of dioc 63 staining as previously described marchetti et al. Since, by convention, the potential outside the cell is defined as zero, the resting potential vr is equal to vin its usual range in neurons is60 my to 70 my. In general, tmrm concentrations range from 20200 nm, and the cell incubation time with. Mitoid membrane potential detection kit the mitoid membrane potential detection kit donated by enzo life sciences, farmingdale, ny was used to assess the change in mmp in perturbed hela cells.
1506 399 296 11 1009 317 409 515 323 535 645 84 1511 1376 177 664 1429 1174 81 1319 1106 865 178 478 1025 1217 1034 225 871 684 527 1251 411 545 1037 589