Optical imaging of cell membrane potential changes induced by applied electric fields

Gross, David J.; Loew, Leslie M.; Webb, W. W.

doi:10.1016/s0006-3495(86)83467-1

Cited by 264 publications

(146 citation statements)

References 41 publications

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“…In contrast, measurements by means of potentiometric dyes are noninvasive (i.e., no physical disruption of the membrane occurs), they offer higher spatial resolution than microelectrodes, and their presence does not distort the electric field. As a consequence, potentiometric dyes, such as di-8-ANEPPS [49], [50], RH292 [31], and ANNINE-6 [51], have become established tools for measurements of ITV, experimental studies of voltage-gated membrane channels, and monitoring of nerve and muscle cell activity. These dyes are incorporated into the lipid bilayer of the cell plasma membrane, where they start to fluoresce, with their fluorescence spectra being dependent on the amplitude of the ITV ( Figure 7A).…”

Section: B) Numerical Computationmentioning

confidence: 99%

Cell membrane electroporation- Part 1: The phenomenon

Kotnik

Kramar

Pucihar

et al. 2012

IEEE Electr. Insul. Mag.

407

212

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Section: B) Numerical Computationmentioning

confidence: 99%

Cell membrane electroporation- Part 1: The phenomenon

Kotnik

Kramar

Pucihar

et al. 2012

IEEE Electr. Insul. Mag.

407

212

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“…For example, for a spherical cell, ΔΦ is given by the Schwan s equation, which states that the voltage is proportional to the field strength and cell size and follows the cosine function along the membrane [8,9]. For more complicated cell shapes, ΔΦ can deviate considerably from the cosine and must be determined either numerically, using a computer [2,10,11], or experimentally, using a potentiometric dye [12,13,14,15].…”

Section: Discussionmentioning

confidence: 99%

“…its resting component, although such efforts were also reported [17]. It is, however, suitable for measuring larger changes in membrane voltage, such as the onset of induced membrane voltage in nonexcitable cells exposed to external electric fields [12,13], or action potentials in excitable cells [4,5]. Although not applied here, di-8-ANEPPS also allows determination of ΔΦ by ratiometric measurements of fluorescence excitation [18] or emission [19], which increases the sensitivity of the response and reduces the abovementioned effects.…”

Section: Discussionmentioning

confidence: 99%

Measuring the Induced Membrane Voltage with Di-8-ANEPPS

Pucihar

Kotnik

Miklavčič

2009

JoVE

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Placement of a cell into an external electric field causes a local charge redistribution inside and outside of the cell in the vicinity of the cell membrane, resulting in a voltage across the membrane. This voltage, termed the induced membrane voltage (also induced transmembrane voltage, or induced transmembrane potential difference) and denoted by ΔΦ, exists only as long as the external field is present. If the resting voltage is present on the membrane, the induced voltage superimposes (adds) onto it. By using one of the potentiometric fluorescent dyes, such as di-8-ANEPPS, it is possible to observe the variations of ΔΦ on the cell membrane and to measure its value noninvasively. di-8-ANEPPS becomes strongly fluorescent when bound to the lipid bilayer of the cell membrane, with the change of the fluorescence intensity proportional to the change of ΔΦ. This video shows the protocol for measuring ΔΦ using di-8-ANEPPS and also demonstrates the influence of cell shape on the amplitude and spatial distribution of ΔΦ. Video LinkThe video component of this article can be found at http://www.jove.com/video/1659/ ProtocolPart I: Preliminary steps 1. In this experiment Chinese hamster ovary cell line (CHO-K1) is used. Cells are plated in Lab-Tek II chambers (2 wells, 4 cm 2 each) (Nalge Nunc, Germany) at ~0.7x10 5 cells/ml in HAM-F12 culture medium supplemented with 8% fetal calf serum, 0.15 mg/ml L-glutamine, 16 mg/ ml gentamicin (all from Sigma-Aldrich, Steinheim, Germany), and 200 units/ml Crystacillin (Pliva, Zagreb, Croatia), and incubated in 5% CO 2 at 37°C. Alternatively, cells can also be plated on #1 glass cover slips (0.13 to 0.16 mm thick), coated with a cellular adhesive such as polylysine. 2. Incubate the cells in their culture medium. Incubation lasting 2 to 4 hours yields cells that are still roughly spherical, but firmly attached to the surface with a small part of their membrane. Alternatively, after 16 to 20 hours of incubation, cells are fully attached to the surface and have more complex shapes, but most of them are still not dividing. 3. Prepare a 10 mM stock solution of di-8-ANEPPS (Invitrogen, Eugene, Oregon, USA) by adding 843 μl of DMSO (Sigma-Aldrich, Steinheim, Germany) to 5 mg of the dye in the original Invitrogen vial. The stock solution can be stored in a refrigerator at 4°C for several months. Before starting the experiments, warm up the the solution until the crystals of DMSO dissolve. 4. Some cell lines may require the use of pluronic to ease the dye incorporation into the cell membrane. Pluronic can be purchased in 20% stock solution in DMSO (F-127, Invitrogen, Eugene, Oregon, USA), or a stock solution of the same concentration can be prepared by dissolving pluronic in DMSO. Stock solution of pluronic can be stored at room temperature. Part II: Loading the cells with di-8-ANEPPS1. Mix 3 μl of 10 mM di-8-ANEPPS and 2.5 μl of 20% pluronic in 1 ml of the Spinner modification (calcium-depleted version) of the Minimum Essential Medium SMEM (medium M8167 or M4767, Sigma-Aldrich, Stein...

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“…These physical predictions were checked experimentally by videomicroscopy by using the potential difference sensitive fluorescent probes. [19][20][21] The pulse duration plays a critical role when shorter than the capacitive loading time of the membrane. In the previous part of the paper, it was considered that the pulse was long enough to bring the potential steady state value.…”

Section: Theory Of Membrane Potential Difference Modulationmentioning

confidence: 99%

“…42 No plasmid membrane interaction occurs if the nucleic acids are added after electropermeabilizing cells as proposed in. 19,38 Negatively charged DNA molecules migrate when submitted to an electric field. 43,46 But, electrophoretic DNA accumulation by itself is not enough to on the electric field parameters.…”

Section: Events During Electropulsation: Membrane -Dna Interactionmentioning

confidence: 99%

Time dependence of electric field effects on cell membranes. A review for a critical selection of pulse duration for therapeutical applications

Teissié¹,

Escoffre²,

Rols³

et al. 2008

Radiology and Oncology

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Background. Electropulsation is one of the non-viral methods successfully used to transfer drugs and genes into living cells in vitro as in vivo. This approach shows promise in field of gene and cellular therapies. This presentation first describes the temporal factors controlling electropermeabilization to small molecules (< 4kDa) and then the processes supporting DNA transfer in vitro. The description of in vitro events brings our attention on the processes occurring before (s), during (ms) and after electropulsation (ms to hours) ofhas the main advantages of being easy to use, fast, reproducible and safe.While during 30 years due to technological limits, pulse duration was always larger than 1 microsecond, the recent availability of high voltage (tens of kV) nanosecond long pulse generators opens the way to a new approach. Very fast perturbations under strong fields are induced in the membrane organization. 16,17 A new field of development is now present for electropermeabilization and promising results for clinical applications were reported.One of the limiting problems remains that very few is known on the physicochemical mechanisms supporting the reorganisation of the cell membrane. The molecular target of the field effect remains unclear.The present review focuses on the critical role played by the pulse duration in the electropermeabilization to small molecules (< 4kDa) and on its support to the processes associated to DNA transfer in vitro. Pulse durations are easy to adjust for an optimization of the clinical target: electrochemotherapy, irreversible electropermeabilization or gene therapy as suggested as a final conclusion. Electropermeabilization Theory of membrane potential difference modulation.An external electric field modulates the membrane potential difference. 18 From the physical point of view, a cell can be described as a spherical capacitor which is charged by the external electrical field. The transmembrane potential difference induced by the electric field, ΔΨi is a complex function g(λ) of the specific conductivities of the membrane (λ m ), the pulsing buffer (λ out ) and the cytoplasm (λ cyt ), the membrane thickness and the cell size. Thus, 1 ΔΨi = f. g (λ). r. E.cosθ [1] in which θ designates the angle between the direction of the normal to the membrane at the considered point on the cell surface and the field direction, E the field intensity, r the radius of the cell and f, which is a shape factor (a cell being a spheroid). Therefore, ΔΨi is not uniform on the cell surface. It is maximum at the positions of the cell facing the electrodes. These physical predictions were checked experimentally by videomicroscopy by using the potential difference sensitive fluorescent probes. [19][20][21] The pulse duration plays a critical role when shorter than the capacitive loading time of the membrane. In the previous part of the paper, it was considered that the pulse was long enough to bring the potential steady state value. The loading time τ load brings a limit in this description. 1 ΔΨi = f. ...

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Optical imaging of cell membrane potential changes induced by applied electric fields

Cited by 264 publications

References 41 publications

Cell membrane electroporation- Part 1: The phenomenon

Cell membrane electroporation- Part 1: The phenomenon

Measuring the Induced Membrane Voltage with Di-8-ANEPPS

Time dependence of electric field effects on cell membranes. A review for a critical selection of pulse duration for therapeutical applications

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