To begin, pipette 1.5 milliliters of the 0.1 milligram per milliliter propidium iodide solution into each well in the electrode array. Place an insert into each well in the electrode array, fitting the feet of the insert into the alignment grooves so the insert is flush with the upper surface of the well. Screw the top electrode printed circuit board to the top of the electrode array wells and connect the electrode array to the porous substrate electroporation, or PSEP device.
Place the electrode array in the 37 degrees Celsius incubator for temperature equilibration. Click the dropdown next to membrane in the top left corner of the GUI and click on 400 nanometer GBO. On the right side of the GUI, type one into the post pulse time duration minute edit field.
Now, click on the run button and enter appropriate names for wells one to three and four to six when prompted. Then, click on okay to start the experiment. After one hour, remove the electrode array from the incubator and transfer the inserts back into the original locations in the experiment plate.
Mix two microliters of Hoechst 33342 and five microliters of calcein AM with 123 microliters of cell culture media in a 200 microliter tube. Gently pipette 10 microliters of the stain solution into each post pulse insert and place the inserts back into the incubator for five minutes. Transfer the well plate to the plate holder of a fluorescent microscope with a 5x magnification objective.
Image using brightfield and the fluorescence of each stain. Pipette 1.5 milliliters of the cell culture media into each well in the electrode array. Place cell sample inserts into wells one to three and control inserts into wells four to six, fitting the feet of the insert into the alignment grooves.
Screw the top electrode printed circuit board to the top of the electrode array wells and connect the electrode array to the PSEP device. Place the electrode array in the 37 degrees Celsius incubator for temperature equilibration. Click on the dropdown next to membrane in the top left corner of the GUI and click on 400 nanometer GBO.
Now, click on the run button and enter appropriate names for wells one to three and four to six when prompted. Then, click on okay to start the experiment. After one hour, remove the electrode array from the incubator and transfer the inserts back into the original locations in the experiment well plate.
After preparing Hoechst 33342, calcein AM, and propidium iodide in cell culture media, mix and gently pipette 10 microliters of the stain solution into each post pulse insert, and place the inserts back into the incubator for five minutes. On a fluorescent microscope, image the cell sample inserts using brightfield and the fluorescence of each stain. The optimized healthy curve showed no dip below the baseline and the largest increase in TEEI.
Post PSEP imaging of the cell monolayer revealed negligible cell death and a healthy fully confluent cell monolayer. The optimized unhealthy data resulted in a diminished TEEI response. Post PSEP imaging revealed near zero cell death and reduced delivery efficiency due to fewer cells in the monolayer.
Application of unoptimized waveforms resulted in significant decreases in TEEI response, indicating substantial cell death and diminished delivery efficiency.
