To begin, use a multi-channel pipette to pipette 100 microliters of 10 millimolar cysteine into the wells of a 96-well-plate in a sterile hood. After incubating the plate, carefully aspirate the solution from the well and avoid scratching the electrode. Rinse the wells two times with sterile deionized water.
Next, add 100 microliters of freshly prepared rat-tail collagen one solution into each well. Well incubate the plate for one hour under low light before washing twice with deionized water. Next, transfer the harvested brain endothelial cells into a fresh sterile trough.
With a multichannel pipette, add 100 microliters of the cell suspension into each well completing seeding in 30 seconds, keep one well media only for mathematical modeling purposes. On a 96-well-plate adapter, move the red clips on each side outward to allow the plate to be inserted. Precisely, align the A one well of the plate with the A one region of the adapter and apply gentle pressure on top of the plate to hold it securely in place.
Then lock the red clips back. Press set up to initiate the instrument after closing the incubator, all correctly detected wells will be indicated by a green color on the plate map. Press the check button to authenticate the absolute impedance readouts.
Now use the dropdown menu beneath the plate map to select the type of plate being used for the experiment. Select multi-frequency to measure the impedance at various alternating current frequencies. Finally, click on the start button to begin the experiment.
Allow the cells to proliferate until a high resistance monolayer is formed indicated by an increase in ohms. Ensure the cell growth has reached a plateau before preparing treatment solutions. To prepare treatment solutions, place labeled 1.1 milliliter polypropylene cluster tubes into strip tube plates.
Add three 50 microliters of the treatment cytokines or cells into the tube following a pre-made plate map. Then place them in the incubator for warmth. On the EISs software.
Click on pause to temporarily halt the ongoing experiment. When prompted, open the incubator and carefully release both red clips while holding the plate steady. With the experiment still in paused state, transfer the plate to the sterile hood.
Then remove the treatment stripped tubes from the incubator. With a multi-channel pipette. Carefully resuspend the contents of the treatment stripped tubes.
Now pipette out 100 microliters of treatment from the stripped tubes and transfer it to the appropriate wells on the 96 well plate, add only complete media to the cell-free wells. Try to finish pipetting in under five minutes as excessive cooling of the plate could affect the resistance of the endothelial cell monolayer. Reattach the treated plate to the machine, then check to assess the electrode well impedance.
Confirm the correct multi-frequency acquisition settings and plate catalog have been selected. Then press resume to continue the experiment. Once the experiment has progressed to the desired endpoint, press finish to automatically save the recorded file.
Navigate to file, and click on export data to export the data as an XLS or CSV file for further analysis. Increase in resistance was observed over 30 hours indicating the growth phase of the endothelial cells. During growth phase, the cells plateaued at different levels by 48 hours, measurement normalization allowed a more reliable interpretation of resistance changes.
Incorrect cell count of the brain endothelial cells before treatment resulted in a fluctuating growth phase, which gradually declined into a stable resistance plateau. Optimized cell seeding density and loading volume resulted in an optimal growth phase. Cytokines appeared to have a transient effect on the barrier.
The addition of melanoma cells drastically reduced the barrier resistance. The paracellular barrier and basolateral component fluctuated with the addition of the cytokines, addition of melanoma cells resulted in a larger magnitude of decrease of the paracellular barrier relative to the basolateral component.
