Assessing Transendothelial Electrical Resistance in an In Vitro Blood-Brain Barrier Model

Abstract

Source: Kuzmanov, I., et al. An In Vitro Model of the Blood-brain Barrier Using Impedance Spectroscopy: A Focus on T Cell-endothelial Cell Interaction. J. Vis. Exp. (2016)

This video demonstrates a method for assessing transendothelial electrical resistance (TEER) in mouse brain microvascular endothelial cells in vitro. A high TEER value indicates intact endothelial cell junctions, while the addition of activated T cells lowers the TEER value, reflecting disruption of the junctional barrier.

Protocol

1. Setting Up and Performing Transendothelial Electrical Resistance (TEER) Measurement

1. Place the 24-well module of the transendothelial electrical resistance (TEER) instrument under the laminar flow hood. Remove the lids and place inserts with primary mouse brain microvascular endothelial cells (MBMECs) in the instrument using forceps.
2. Pipette 810 µl of fresh medium to the lower compartment of the module wells: add it carefully between the insert and the wall of the module well.
3. Close the lids and place the instrument in the incubator. Connect the instrument to its computer; turn on the instrument controller and op....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
cellZscope	nanoAnalytics GmbH	www.nanoanalytics.com	including: 24-well Cell Module, Controller, PC with cellZscope software v2.2.2
Transwell membrane inserts - pore size 0.4 µm	Corning	3470	for TEER measurement as the main readout
Transwell membrane inserts - pore size 3 µm	Corning	3472	for TEER measurement as the quality control prior to T-cell transmigration assay
24-well cell culture plate	Greiner	650 180	flat-bottom; for MBMEC culture
96-well cell culture plate	Costar	3526	round-bottom; for immune cell culture
Neubauer counting chamber	Marienfeld	MF-0640010	for cell counting
Collagen type IV from human placenta	Sigma	C5533	for MBMEC coating solution
Fibronectin from bovine plasma	Sigma	F1141-5MG	for MBMEC coating solution
DMEM (+ GlutaMAX)	Gibco	31966-021	for MBMEC isolation and MBMEC culture medium
Penicillin/Streptomycin	Sigma	P4333	for MBMEC isolation and MBMEC culture medium
Heparin	Sigma	H3393	for MBMEC culture medium
Human Basic Fibroblast Growth Factor (bFGF)	PeproTech	100-18B	for MBMEC culture medium
IMDM + 1% L-Glutamin	Gibco	21980-032	for T cell culture medium
Recombinant Murine IFN-γ	PeproTech	315-05	for T-cell transmigration assays
Recombinant Murine TNF-α	PeproTech	315-01A	for T-cell transmigration assays
NA/LE purified anti-mouse IFN-γ antibody	BD Biosciences	554408	clone XMG1.2; recommended final concentration: 20 µg/ml
Granzyme B Inhibitor II	Calbiochem	368055	recommended final concentration: 10 µM
PE anti-mouse CD4 antibody	Biolegend	116005	clone RM4-4; for analysis of T cell transmigration
Trypan Blue solution, 0.4%	Thermo Fisher Scientific	15250061	for cell counting