This protocol successfully records the effects of the aconitine on the IKv of H9C2 cells. Promoting new phases for investigating the mechanism of cardiotoxicity of drugs from the perspective of ion channels. The whole-cell patch-clamp technique is the gold standard of ion channel research with high sensitivity, high precision, and high specificity.
The whole-cell patch-clamp technique can be employed to explore the pharmacological or toxic mechanisms of juxta gating specific ion channels for multisystem diseases, such as cardiovascular system and nervous system. To begin, digest the cells with 0.25%trypsin for 30 seconds, once the H9C2 culture dish becomes 80%confluent. Culture 2 times 10 to the 5th cells per milliliter with normal medium or drug containing medium in a 35 millimeter dish carpeted with glass plates for 24 hours at 37 degrees celsius, with a 5%carbon dioxide atmosphere and 70 to 80%relative humidity.
Turn on the micropipette puller and preheat for 30 minutes. Then put a borosilicate glass capillary with a filament on the micropipette puller. Select the program and click on enter on the control panel.
Click on the ramp program in the upper right corner to determine the heat value of the glass capillary. Write the program for pulling the electrode according to the value determined by the ramp test and click on pull to start fabricating the pipettes. Turn on the digital analog converter, the signal amplifier, the micro manipulator, the microscope, and the camera.
Open the imaging application, the signal amplifier software and the data acquisition software in sequence. Click on acquire and select edit protocol in the data acquisition software. Edit the program in the waveform interface.
Enter values for first level, delta level, first duration, and delta duration for epoch A, epoch B, and epoch C.Then click on the mode rate interface and set the trial hierarchy data. Next, click on edit protocol to select the stimulus button and set the leak subtraction program in the PM leak subtraction dialogue box. To establish the data storage path, click on file and select set data file names in the data acquisition software.
Then open the established lKv protocol by selecting acquire and clicking on open protocol in the data acquisition software. Finally, click on the tools option, and select membrane test to start running the protocol. Add the extracellular solution to the cell bath on the whole-cell patch-clamp apparatus and place the cover slip upward with the H9C2 cells in the bath.
Fill 30%of the pipette with the extracellular solution and install it on the recording electrode holder integrated into the patch-clamp apparatus. Tighten the pipette with the recording electrode holder. Then drop the pipette into the bath with the micro manipulator.
Click on the pipette offset interface of the signal amplifier software to maintain the lKv current baseline at zero picoamps. Deliver an appropriate positive pressure manually with a 1.0 milliliter syringe, connected to the recording electrode holder, through a plastic tube. Move the pipette slightly by manipulating the micromanipulator in three dimensions to contact the cell.
Once the membrane tests square wave drops by one third to a half after the pipette touches the cell membrane, remove the positive pressure and manually deliver an appropriate negative pressure. Then click on the patch interface of the data acquisition software to form the gigaseal. Use the capacitance compensation fast and capacitance compensation slow signal amplifier software during cell ceiling to compensate for the fast and slow capacitance.
Apply brief pulses of negative pressure to rupture a patch of the cell membrane. Execute whole-cell membrane capacitance compensation by clicking on the whole-cell button of the signal amplifier software. Finally, save and record the data, by clicking on the data recording button.
Open the saved lKv data with the data analysis software. Save the current voltage relationships. Click on analyze to select statistics.
Select the range as Cursors 1 2 for data analysis, click on the peak amplitude and mean buttons and then click on okay to view the data in the results page. Finally, copy the column of lKv mean data into the function drawing software for further analysis. Save the representative lKv current traces.
Click on edit to select transfer traces, then select the full trace in the region to transfer. Next, click on select in trace selection and select IN 0 in signals. Finally, click on okay to view the data in the results page and copy the total data into the function drawing software to draw the current traces.
Save the lKv protocol. Click on edit to select create stimulus waveform signal and select okay. Then repeat the step for drawing current traces without selecting the signal.
The lKv was triggered by 150 milliseconds of depolarizing pulse stimulus from minus 40 to plus 60 millivolts at holding potential of minus 60 millivolts. The lKv of the H9C2 rat cardiomyocytes, first appeared around minus 20 millivolts, and then the amplitude increased with further depolarization. In comparison with the control group, the lKv amplitude was observably reduced after the five minutes of treatment with 1.5 millimolar 4-AP.
Additionally, the lKv decreased significantly at membrane potentials from 10 to 60 millivolts in a dose-dependent manner, after the 24 hours AC treatment. The individual cell after recording ion channel, can be used for further single-cell omics assess, such as single-cell genomics, transcriptomics, proteomics, and the metabolomics.
