Measuring TEER in X-ray-Irradiated Epithelial Cells Co-cultured With PBMCs

Take a membrane insert containing an X-ray-irradiated epithelial monolayer.

Place the membrane insert into a well containing peripheral blood mononuclear cells, or PBMCs — immune cells that secrete cytokines, and other signaling molecules.

Place the transepithelial electrical resistance, or TEER electrodes, into the membrane insert.

Apply a small electrical current and measure the resistance to assess monolayer permeability regulated by tight junctions.

Tight junctions are transmembrane proteins linked to the cytoskeleton through scaffold proteins.

Together, these junctional proteins form a robust barrier between cell membranes, restricting the passage of large molecules and pathogens.

Intact junctional proteins impede electrical current flow, resulting in a high TEER, indicating normal monolayer permeability.

X-ray exposure reduces junctional-protein levels, increasing monolayer permeability and lowering TEER.

Moreover, signaling molecules from irradiated cells activate co-cultured  PBMCs that release cytokines, triggering a further reduction in junctional-protein levels and disrupting the barrier integrity.

Consequently, monolayer permeability increases, causing a further decrease in TEER.

To assess the transepithelial electrical resistance, or TEER, of the Caco-2 cells immediately after irradiation, transfer half of the Caco-2 cell culture inserts into a new plate with 3 milliliters of fresh complete medium in each well, and half of the insert cultures into a new plate seeded with 2 x 10⁶ PBMC per 3 milliliters of complete medium per well. Then, place a TEER chopstick electrode into the cell culture insert every hour for the first six hours, and then every three hours until 48 hours after irradiation.