This article describes a method to measure transendothelial electrical resistance (TEER) in a model of the blood-brain barrier using mouse brain microvascular endothelial cells (MBMECs). The interaction of activated T cells with endothelial cells is assessed to understand the disruption of junctional integrity.
Begin with a membrane insert containing mouse brain microvascular endothelial cells, resembling an in vitro blood-brain barrier.
Place this insert into a transendothelial electrical resistance or TEER instrument well containing an embedded electrode.
Add fresh media to the lower compartment.
Place the lid with electrodes over the TEER wells, then put the instrument in an incubator and connect it to a recording system.
The electrodes generate an electrical current across the cell layer for measurement of transendothelial resistance.
Intact junctional proteins between the cells impede the flow of current, resulting in a higher TEER value.
Disconnect the instrument and remove it.
Disassemble the lid, remove some medium from the upper compartment, and add pre-activated T cells. Reassemble the instrument for TEER measurement.
Activated T cells interact with the endothelial cells, releasing inflammatory factors and cytolytic enzymes, disrupting the junctional integrity.
This disruption increases electrical current flow, resulting in a lower TEER value.
To set up TEER measurement, place the 24-well module of the TEER instrument in the laminar flow hood. Remove the lids, and place the inserts with MBMECs in the instrument using forceps.
Next, pipette 810 microliters of the fresh medium to the lower compartment of the module wells. Add it carefully between the insert and the wall of the module well. After that, close the lids and place the instrument in the incubator. Connect the instrument to the computer. Then, turn on the instrument controller and open the software.
In the pop-up window, select 'New measurement'. Check 'Show TEER' and 'Show Ccl' boxes, then press 'Start'. After completing the first measurement, select 'Check all wells' in the 'results' tab to see all TEER and Ccl values. Then save the file.
Next, choose the time point to co-culture MBMECs with T cells when Ccl is stable and lower than 1 microfarad per square centimeter and TEER has reached its maximum level. Carefully inspect the absolute TEER and Ccl values, and exclude the wells in which MBMECs have not developed confluent enough monolayers by unchecking such wells.
Group the rest of the wells by right-clicking on the wells and selecting 'Add well to new average well'. Name it in the pop-up window and do the same for all individual wells to be grouped. Then, check all average wells to confirm that all of them have the same initial conditions before the co-culture.
In the 'Experiment' tab, press 'Pause'. Disconnect the instrument and take it out of the incubator. Then, remove the lids in the laminar flow hood. Subsequently, remove some of the medium from the Insert at the upper well compartment. Add T cells to MBMECs by carefully pipetting 150 microliters of medium. Then, reconnect the instrument, and press 'Resume measurement'. After 24 hours, press 'Stop' in the 'Experiment' tab and save the file.
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