Transepithelial/transendothelial electrical resistance assay
Materials Required
Principle
TEER measures electrical resistance across epithelial or endothelial monolayers cultured on permeable supports, and the readout reflects ionic conductance through the cell barrier, especially the paracellular pathway regulated by junctional integrity[1][2][3]. TEER can be measured without destroying the monolayer and is commonly used before or during transport, permeability, barrier-disruption, and barrier-maturation experiments[1][2][3]. TEER values are influenced by biological maturation and technical conditions; reported factors include temperature, medium formulation, passage number, electrode geometry, membrane properties, and junctional length during early monolayer maturation[1][3][5]. Therefore, TEER should be interpreted with blank-insert subtraction, area normalization, repeated readings, and, when possible, orthogonal barrier readouts such as FITC-dextran flux or tight-junction staining[4][5][6].
MCE has not independently verified the accuracy of these methods. They are for reference only.
Experimental Materials
• Use barrier-forming epithelial or endothelial cells, complete culture medium appropriate for the selected model, permeable Transwell-style inserts, PBS or culture medium for handling, and cell-free inserts for blank resistance subtraction[1][3][4][6].
• For a Caco-2 example, published studies used DMEM supplemented with serum and antibiotics, seeded cells on Transwell inserts, and cultured monolayers until barrier formation before TEER measurement[4][6].
• FITC-dextran can be used as a complementary paracellular permeability tracer, and tight-junction markers such as ZO-1, occludin, and claudin-1 can be assessed by immunofluorescence or protein-expression assays to cross-check TEER-based barrier interpretation[4][5][6].
• Use a TEER meter or impedance system with compatible electrodes, permeable inserts, sterile cell-culture plates, CO2 incubator, biosafety cabinet, and standard cell-culture equipment[1][2][3][4].
• Chopstick-style electrodes, EndOhm-style chambers, impedance systems, and integrated organ-chip electrodes have all been reported, but electrode geometry and device design can affect measured resistance[1][2][3].
• For a Caco-2 example, published studies used DMEM supplemented with serum and antibiotics, seeded cells on Transwell inserts, and cultured monolayers until barrier formation before TEER measurement[4][6].
• FITC-dextran can be used as a complementary paracellular permeability tracer, and tight-junction markers such as ZO-1, occludin, and claudin-1 can be assessed by immunofluorescence or protein-expression assays to cross-check TEER-based barrier interpretation[4][5][6].
• Use a TEER meter or impedance system with compatible electrodes, permeable inserts, sterile cell-culture plates, CO2 incubator, biosafety cabinet, and standard cell-culture equipment[1][2][3][4].
• Chopstick-style electrodes, EndOhm-style chambers, impedance systems, and integrated organ-chip electrodes have all been reported, but electrode geometry and device design can affect measured resistance[1][2][3].
Experimental Procedure
• Seed cells on permeable inserts at a density supported by the chosen model and culture until a stable barrier is formed; in Caco-2 examples, cells were seeded on 12-well inserts at 2 × 105 cells/well or 6-well inserts at 106 cells/well and cultured for 21 days, while another study compared TEER across 15, 18, 21, and 24 days of Caco-2 culture[4][6].
• Replace medium according to the selected literature-supported model; one Caco-2 study changed medium every other day in week 1 and daily during the next 2 weeks, while another TEER study exchanged medium every 2 days[4][5].
• Prepare a cell-free insert containing the same medium as the experimental wells to measure blank resistance, because published TEER calculations subtract the blank insert value before multiplying by membrane area[4][5].
• Maintain comparable measurement conditions across groups because TEER is affected by physical and technical variables, including temperature, medium composition, membrane features, and electrode configuration[1][3][5].
• Measure TEER after the monolayer reaches the predefined model-specific readiness criterion; in one Caco-2 barrier-disruption study, only monolayers above 1,000 Ω·cm2 were used for subsequent treatment, while broader literature shows that absolute TEER values vary substantially across cell models and technical setups[1][4][5][6].
• Place the electrodes according to the instrument configuration so that resistance is measured across the apical and basolateral compartments without damaging the monolayer[1][3][4].
• Record each well multiple times and average the readings; one Caco-2 study measured each well three times and used the average resistance value[4].
• Calculate area-normalized TEER as: TEER = (measured resistance − blank resistance) × membrane area, reported as Ω·cm2[4][5].
• For barrier-disruption or protection experiments, apply treatment after barrier formation and measure TEER at literature-supported time points; one Caco-2 study treated monolayers with 25 μg/mL Trichinella spiralis excretory-secretory products or 20 ng/mL TNF-α for 48 h, using medium as a negative control and TNF-α as a positive barrier-disruption control[4].
• When using FITC-dextran as an orthogonal permeability assay, one study replaced medium with serum-free DMEM for 30 min at 37°C and measured apical-to-basolateral 4 kDa FITC-dextran flux for 1 h[4].
• Report raw resistance, blank-subtracted resistance, membrane area, and final Ω·cm2 values, because TEER comparisons require blank subtraction and area normalization[1][4][5].
• Analyze TEER as relative change from baseline or matched untreated controls when comparing treatments, because absolute TEER values differ by cell type, culture maturity, medium, membrane, and electrode system[1][3][5][6].
• Interpret decreased TEER as evidence of increased ionic conductance across the barrier only in the context of controls and complementary assays; Caco-2 barrier disruption was supported by concurrent TEER decrease, increased FITC-dextran flux, and altered tight-junction markers[4][5].
• Use biological replicates and report dispersion; one Caco-2 study analyzed three independent experiments and used Student’s t-test or one-way ANOVA for TEER and fluorescence data[4].
• Replace medium according to the selected literature-supported model; one Caco-2 study changed medium every other day in week 1 and daily during the next 2 weeks, while another TEER study exchanged medium every 2 days[4][5].
• Prepare a cell-free insert containing the same medium as the experimental wells to measure blank resistance, because published TEER calculations subtract the blank insert value before multiplying by membrane area[4][5].
• Maintain comparable measurement conditions across groups because TEER is affected by physical and technical variables, including temperature, medium composition, membrane features, and electrode configuration[1][3][5].
• Measure TEER after the monolayer reaches the predefined model-specific readiness criterion; in one Caco-2 barrier-disruption study, only monolayers above 1,000 Ω·cm2 were used for subsequent treatment, while broader literature shows that absolute TEER values vary substantially across cell models and technical setups[1][4][5][6].
• Place the electrodes according to the instrument configuration so that resistance is measured across the apical and basolateral compartments without damaging the monolayer[1][3][4].
• Record each well multiple times and average the readings; one Caco-2 study measured each well three times and used the average resistance value[4].
• Calculate area-normalized TEER as: TEER = (measured resistance − blank resistance) × membrane area, reported as Ω·cm2[4][5].
• For barrier-disruption or protection experiments, apply treatment after barrier formation and measure TEER at literature-supported time points; one Caco-2 study treated monolayers with 25 μg/mL Trichinella spiralis excretory-secretory products or 20 ng/mL TNF-α for 48 h, using medium as a negative control and TNF-α as a positive barrier-disruption control[4].
• When using FITC-dextran as an orthogonal permeability assay, one study replaced medium with serum-free DMEM for 30 min at 37°C and measured apical-to-basolateral 4 kDa FITC-dextran flux for 1 h[4].
• Report raw resistance, blank-subtracted resistance, membrane area, and final Ω·cm2 values, because TEER comparisons require blank subtraction and area normalization[1][4][5].
• Analyze TEER as relative change from baseline or matched untreated controls when comparing treatments, because absolute TEER values differ by cell type, culture maturity, medium, membrane, and electrode system[1][3][5][6].
• Interpret decreased TEER as evidence of increased ionic conductance across the barrier only in the context of controls and complementary assays; Caco-2 barrier disruption was supported by concurrent TEER decrease, increased FITC-dextran flux, and altered tight-junction markers[4][5].
• Use biological replicates and report dispersion; one Caco-2 study analyzed three independent experiments and used Student’s t-test or one-way ANOVA for TEER and fluorescence data[4].
Troubleshooting
Problem: TEER is low or unstable after apparent confluence.
• Possible cause: The monolayer may be immature, because TEER during early Caco-2 maturation can be affected by changing cell number, junctional length, and incomplete junctional maturation.• Literature-supported solution: Continue model-specific maturation and confirm barrier status with tight-junction staining or FITC-dextran permeability rather than relying only on confluence[5].
Problem: Similar monolayers show different absolute TEER values.
• Possible cause: TEER is affected by technical factors such as temperature, medium formulation, membrane properties, and electrode configuration.• Literature-supported solution: Keep measurement conditions consistent, subtract blank-insert resistance, normalize by membrane area, and avoid comparing absolute TEER values across different devices or culture formats without validation[1][3][5].
Problem: TEER changes do not match FITC-dextran permeability.
• Possible cause: TEER reflects ionic conductance, whereas FITC-dextran flux reflects macromolecular paracellular permeability, so the two readouts can diverge during monolayer maturation.• Literature-supported solution: Interpret TEER with a complementary permeability assay and junctional-marker assessment when barrier status is uncertain[4][5].
References:
- [1]. Srinivasan B, et al. TEER measurement techniques for in vitro barrier model systems. J Lab Autom. 2015;20(2):107-126. [Content Brief]
- [2]. Henry OYF, et al. Organs-on-chips with integrated electrodes for trans-epithelial electrical resistance (TEER) measurements of human epithelial barrier function. Lab Chip. 2017;17(13):2264-2271. [Content Brief]
- [3]. Vigh JP, Kincses A, Ozgür B, Walter FR, Santa-Maria AR, Valkai S, et al. Transendothelial electrical resistance measurement across the blood-brain barrier: a critical review of methods. Micromachines (Basel). 2021;12(6):685. [Content Brief]
- [4]. Li C, Bai X, Liu X, Zhang Y, Liu L, Zhang L, et al. Disruption of epithelial barrier of Caco-2 cell monolayers by excretory secretory products of Trichinella spiralis might be related to serine protease. Front Microbiol. 2021;12:634185. [Content Brief]
- [5]. Felix K, et al. Measurements of transepithelial electrical resistance (TEER) are affected by junctional length in immature epithelial monolayers. Histochem Cell Biol. 2021;156(6):609-616. [Content Brief]
- [6]. Lopez-Escalera S, et al. Evaluation of Caco-2 and human intestinal epithelial cells as in vitro models of colonic and small intestinal integrity. Biochem Biophys Rep. 2022;31:101314. [Content Brief]
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