This article describes a method for recording light-induced ion currents in embryonic kidney cells using patch-clamp techniques. The process involves isolating transfected cells and applying specific light wavelengths to activate ion channels.
Place a coverslip containing embryonic kidney cells expressing light-dependent ion channels and a fluorescent reporter protein on a silicon-applied measuring chamber. Close it and add an external buffer.
Position this chamber under a microscope. Place a bath electrode filled with agar. Perfuse with a buffer to remove residual media and dead cells.
Using a fluorescence filter, identify and focus on the target cell.
Attach a patch pipette to a micromanipulator. Apply pressure to prevent clogging and approach the cell.
Position the pipette near the cell and release positive pressure to form a membrane patch at the tip.
After forming the membrane patch, apply negative pressure to rupture the membrane.
Apply a light of a specific wavelength to open the ion channels. The movement of ions is recorded as voltage change by the electrode.
To begin, seal the measuring chamber with silicon to prevent leakage of the external buffer. Then, place one coverslip into the chamber and close it. Fill the chamber carefully with extracellular buffer to prevent the cells from detaching. Then, place the measuring chamber under the microscope using the 40x objective for cell visualization.
Next, put an agar bridge over the bath electrode and place it into the chamber together with the fluid level sensor and the perfusion outlet of the bath handler. Exchange the extracellular solution twice with 1 milliliter of fresh external solution to remove the residual culture medium and the detached cells. Bring the cells into focus and search for a transfected cell, which needs to be isolated from other cells. Then, use a triple band filter set and orange light to excite and visualize mCherry.
Mount the pipette on the pipette holder, and apply some positive pressure to prevent tip clogging. Locate the tip of the patch pipette under the microscope and navigate it close to the cell using the micromanipulator.
Within the data acquisition software, start the membrane test in Bath mode and apply a voltage step. Check if the pipette resistance is in the desired range of 1.3 to 3.0 megaohms. Then, zero the offset currents and adjust the pipette potential by turning the pipette offset knob on the amplifier.
To establish a patch in the whole-cell configuration, slowly approach the cell with the patch pipette from the top and release the positive pressure right before touching the cell. Compensate the pipette capacitance by turning the pipette capacitance compensation knob to get a flat response of the test pulse.
Switch the membrane test to Cell mode. Then, rupture the patch without destroying the seal by applying short pulses of negative pressure or negative pressure with increasing strength to obtain whole-cell conformation.
Start series resistance compensation by setting the two whole-cell parameters, cell capacitance and series resistance. Record light-induced currents at different holding potentials. This figure shows that during the illumination with green light, PSACR1 features a fast transient current which rapidly decays to a stationary current level.
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