Amphotericin-B Mediated Perforated Patch-Clamp Analysis: An Electrophysiological Technique to Study Ionic Currents in Urinary Bladder Cells
Transcripción
Pipette 0.25 to 1 milliliter of cell suspension onto a glass-bottom chamber sitting on the stage of an inverted microscope and incubate it for at least 45 minutes to allow the cells to adhere. Then, remove the DS from the bath and replace it with E solution via superfusion.
Pull multiple patch electrodes, fire-polish the electrode tips, and coat the tips in dental wax if needed. Fill the tip of a patch electrode with the pipette solution without amphotericin-B by briefly dipping the electrode in the solution. Success of patch-clamp electrophysiology depends on DSM cell quality and amphotericin-B solubilization.
Backfill the electrode with the same pipette solution containing amphotericin-B and mount the electrode onto a holder connected to a patch-clamp amplifier head stage. Use a micromanipulator to place the electrode just below the surface of the extracellular solution so that the tip of the electrode is just submerged.
Then, determine the electrode resistance using the Membrane Test window function of the commercial acquisition software and advance the electrode toward the cell of interest. When touching the cell surface with the electrode, form a giga-seal by applying gentle rapid negative pressure to the electrode via tubing. This results in negative pressure at the tip of the electrode, resulting in a successful giga-seal as confirmed by the Membrane Test.
Allow 30 to 60 minutes for the amphotericin-B to diffuse down the pipette and be inserted into the plasma membrane, forming pores primarily selective to monovalent cations. Continue monitoring the giga-seal with the Membrane Test function.
When the patch perforation is optimal, cancel out the capacitance transients by adjusting the dials for cell capacitance and series resistance on the amplifier. Once the stable voltage-step cation currents are observed, record currents with the routing voltage-step protocol as described in the manuscript.
