Cut off both eyes using the razor blade chip by using a sawing motion along the line of the frontal margin of the eye. Figure 4: Table 1: Offset the capacitive currents of the pipette by adjusting the appropriate knob in the patch clamp amplifier.
For detailed description of series resistance compensation and correction refer to the amplifier manual or guideline as this procedure varies among different manufactures. Firmly grasp the edge of one cornea with the fine tweezers and scoop out the retina using the scooper. Prepare all solutions according to the instructions in Tables 1-4. The setup contains an iron, black-painted Faraday cage F.
At the first stage, the surrounding pigmented glia should disintegrate, leaving visible small debris in the solution. Incubate the retinae in the dark for 20-25 min. This allows the cell content to equilibrate with the pipette solution.
Table 2: Your institution must subscribe to JoVE's Neuroscience section to access this content. Carefully fill the chamber with extracellular buffer high [Cl - ] to prevent detaching of cells, then place it under the microscope and use the 40X objective for cell visualization.
Recovery current values are obtained by dividing the peak current from P2 by the peak current at P1. Patch-clamp Setup.HEKA Electrophysiology Update 2013-12-18
Close the tube valve to maintain the pressure. Open the "membrane test module" to apply continuous square voltage pulses of 2 mV at a rate of 100 Hz.
This can be done by using either a syringe, via a mouth piece or an electronically controlled pressure regulator. This technique allows control over the intracellular and extracellular media; the membrane voltage; and the fast application of pharmacological compounds, such as a variety of ionic or pH indicators, to the intra- and extracellular media. Print ISSN: Abstract This protocol is used to measure sodium currents from heterologously transfected cells lines, such as HEK293 cells.
Later, the addition of ATP and NAD to the patch pipette dramatically increased the suitability of the preparation for prolonged quantitative recordings. Use any suitable homemade or commercial chamber that allows electrode access and perfusion. PLoS One 8 1: Zero offset currents by adjusting the pipette potential by turning the pipette offset knob at the amplifier while working in "bath mode" of the "membrane test".
Determination of reversal potentials at defined ionic and electrical conditions provides information about the ion species transported after light activation of ChRs.