Measuring the Induced Membrane Voltage with Di-8-ANEPPS

For a biologist, a cell is the building brick of life containing a number of different organelles, each accomplishing its specific tasks and together allowing the cell to perform its vital functions from an electrical perspective. However, the internal structure is not very important, and the cell can be viewed as a conductive cytoplasm enclosed by an insulating membrane and surrounded by a conductive exterior. A small imbalance in ionic concentrations with the surplus ions accumulating close to the membrane results in the resting membrane voltage.

Placing a cell into an electric field causes a local charge redistribution on both sides of the membrane. The result is the induced membrane voltage, which superposes to the resting voltage and disappears after the field is turned off. With simple cell shapes, the induced membrane voltage can be calculated analytically for a spherical cell.

It is given by the schwan's equation, which states that the voltage is proportional to the field strength and cell size, and follows the co-sign function along the membrane. For more complicated cell shapes, the induced voltage can deviate considerably from the cosign function and must be determined either numerically using a powerful computer or experimentally using a potential metric dye. One of the potential metric dyes widely used for this purpose is diet AIPs.

A fast dye with excitation and emission spectra dependent on the membrane voltage diet, AIPs becomes strongly fluorescent when bound to the membrane with the change of the fluorescence intensity proportional to the change of the membrane voltage. This video shows the protocol for measuring the induced membrane voltage by using D AIPs. In this Example, China's hamster ovary cells are used plate the cells in chambers at 100, 000 cells per chamber.

In HAMF 12 culture, medium supplemented with fetal cal serum L glutamine, and antibiotics incubate the cells in their culture medium for two to four hours to obtain attached cells of spherical shape, or 16 to 20 hours to obtain single attached cells of more complex shapes. Prepare 10 million MO stock solution of diet aaps dissolved in DMSO in the original vial. Some cell lines may require additional of onic to ease the dye incorporation into the membrane Transfer one milliliter of spinners, modification of the minimum essential medium SMEM into an EOL tube at 2.5 microliters of 20%onic and three microliters of 10 millimolar diet aaps.

This yields a loading solution containing approximately 30 micromolar diet aaps and 0.05%of onic. Replace the culture medium in the chamber with the loading solution. Transfer chamber to the refrigerator for 10 minutes at four degrees Celsius at this temperature.

Internalization of the dye through the plasma membrane is largely inhibited after staining, carefully wash the access dye several times with pure SMEM. Finally, leave 1.5 milliliters of SMEM in the chamber. The cells are observed using a fluorescence microscope with an oil immersion objective, a monochromator and a CCD camera.

In the acquisition software, set the excitation wavelength to 419 nanometers and chose a band pass filter sent at roughly 605 nanometers, although not applied here. Diet aaps also allows ratio metric measurements of membrane voltage. Place the chamber with cells onto the microscope stage, position the electrodes at the bottom of the chamber and connect them to the pulse generator.

In this example, a square pulse with 35 volts amplitude and 50 millisecond duration is generated. Using a DC voltage supply and a microprocessor controlled switcher, the pulse is delivered to a pair of electrodes, creating an electric field between them and inducing the membrane voltage. The pulse delivery must be synchronized with the image inquisition.

Find the cells of interest, apply a single electric pulse or a sequence of pulses for each pulse. Acquire two fluorescence images, the control image immediately before the pulse and the pulse image during the pulse due to the low response of the dye. The changes in the fluorescence are hard to distinguish and become apparent only After processing open the images for each pulse.

Subtract the background in both the control image and the pulse image. Choose a cell and set the region of interest so that it corresponds to the membrane. Measure the fluorescence along this region in the control and pulse image and transfer the values to a spreadsheet for each pulse.

Subtract the control data from the pulse data and divide the result by control data to obtain the relative fluorescence changes. If a sequence of pulse is applied, the relative change values determined for each pulse can be averaged. To get a more reliable measurement, transform the relative fluorescence changes into the induced membrane voltage.

Using a calibration curve, a rough estimation of this curve can be obtained from the literature, but for higher accuracy, it has to be measured for each particular setup. Plot the voltage as a function of the relative arc length. The curve can also be smooth using a suitable filter for spherical cells.

The resulting plot is close to a co-sign in agreement with the schwan's equation. For more complicated cell shapes, the same procedure yields more intricate spatial distributions of the membrane voltage.