To begin, in the sterile hood, screw autoclave 24 well pots into the ethanol sterilized cell module. Add 900 microliters of base media into each pot. Now connect the autoclave dipping electrodes magnetically to the cell module lid.
Then close the lid over the wells so that the dipping electrodes fit into the bottom electrode pots. Next, attach the cell module to the adapter in an incubator. Then launch the software, and click on the layout tab to load and annotate the experimental plate map.
Use a sterile 24 well culture plate to hold the membrane inserts. Where coating is required, add extracellular matrix solution to the intersection of the well With a single channel pipette, carefully seed the brain endothelial cells into the apical chamber of each well insert. To create a cell-free well, pipette only complete media into one of the inserts.
Then transfer the cell module from the incubator into a sterile hood. Using tweezers, carefully transfer the prepared inserts into the electrode pots. Place the cell module back in the incubator over the adapter.
Then locate the spectrum section in the experiment control tab, and select Start at one hertz, and stop at 100 kilohertz to acquire data over the entire frequency range. Next, under the wait time, select 15 minutes to permit continuous measurements at the fastest rate. Press start to initiate data collection.
After approximately 48 hours, once a plateau in the endothelial resistance is reached, place the prepared treatments in an incubator to maintain an optimal temperature. Remove the cell module at a selected treatment time that begins immediately after a 15 minute measurement. In a sterile hood, open the cell module.
Then carefully pipette 70 microliters of the treatment into the apical chamber of the respective insert. Immediately return the cell module to the adapter before the next measurement begins. To end the experiment, press the stop button, then click on file, followed by export to export the data directly to an XLS file, and name it appropriately.
While some variation was observed between replicate wells, data normalization reduced this variation, and allowed for a better comparison of the treatment with the control. A transient decrease in trans endothelial electrical resistance was observed when cytokines were added. The melanoma cell lines caused a substantial decrease in resistance within five hours.
