The TEER operation SOP for barrier function testing is indispensable

The TEER operation SOP for barrier function testing is indispensable.

The TEER (Trans-Epithelial/Endothelial Electrical Resistance) experiment is a standard method for evaluating the integrity of cell monolayer barriers, the formation of tight junctions, and ion permeability. It is widely used in models such as the blood-brain barrier (BBB experiment), intestinal barrier, and respiratory epithelium. The principle of its operation is to detect the integrity of the barrier formed by cells through Ohm's law. The higher the resistance value, the more complete the tight junctions between cells and the stronger the barrier function. 

The TEER experiment operation procedure is here! 

1. Instrument Preparation:

Epithelial/Endothelial Voltage Ohmmeter: Such as EVOM2/EVOM3 (World Precision Instruments), Millicell ERS 3.0, etc. 

Electrode: 

Chopstick electrodes: Handheld type, suitable for rapid screening 

Cavity electrode (EndOhm chamber): Provides more stable and higher repeatability measurements 

Transwell chamber: Commonly available in 6.5 mm (membrane area 0.33 cm²) or 12 mm specifications, with a pore size of 0.4 μm (the size needs to be selected based on the type of cells) 

Sterile forceps 

II. Reagent Preparation

Complete medium (prepared according to the type of cells) 

PBS without Ca²⁺/Mg²⁺ (used for electrode cleaning) 

Collagen/Matrix Gel/Cell Coating Solution (for adherent cell lines and primary cells that are not well attached) 

III. Initial Cell Culture Setup

1. The density of cell culture plates varies. Cells that grow slowly and those with unstable cell attachment on the plate require an increase in plate density. The normal cell inoculation density is approximately 1*10^5 cells/ml. 

2. In the lower chamber, add 1–1.5 mL of cell-free complete medium first, then take 1 ml of cell suspension and inoculate it into the upper chamber of the Transwell chamber; place it in a 37°C, 5% CO₂ incubator for cultivation. 

3. Once the cells form tight junctions, measure the resistance index once every day until it reaches a plateau. 

Measurement steps: 

Transfer the Transwell chamber using sterile forceps into the EndOhm chamber. 

· Ensure that the liquid volumes in the upper and lower chambers are consistent, eliminating errors caused by the height difference of the liquid surface. 

Measure the resistance value according to the instrument manual. 

IV. Calculating TEER value (unit area resistance)

Since resistance is inversely proportional to the membrane area, the tissue resistance needs to be multiplied by the effective membrane area to standardize it as Ω·cm²: TEER = (Rtotal - Rblank) × A 

Among them:

A = Effective area of Transwell membrane (cm²) 

For example: 6.5 mm Corning Transwell: A = 0.33 cm² 

12 mm Transwell: A = 1.12 cm² 

V. Time of Formation of Partial Cell Barriers 

Primary cells (such as primary brain microvascular endothelial cells): The barrier formation is more complete, but the culture period is longer (10–14 days or longer). If co-cultured with pericytes, a complete barrier can generally be formed within 5-10 days. 

Immortalized cell lines (such as bEnd.3, hCMEC/D3): Rapidly proliferating, usually a detectable barrier can be formed within 5-7 days. 

iPSC differentiated cells: The process of maturation takes 10 to 21 days. 

Caco-2: ≥ 300–500 Ω·cm² (usually requires more than 20 days of cultivation) 

Respiratory epithelium: >500 Ω·cm², maintained for at least 12-15 days 

Six. Pay attention to details and achieve success once: 

The temperature needs to be controlled precisely: for every 1°C change in temperature, the resistance value can vary by approximately 2–3%. It is recommended that all samples be measured at the same temperature or undergo temperature correction. 

The volume of the liquid should be uniform: During each measurement, the volumes of the liquid in the upper and lower chambers must be strictly the same. Any difference in the liquid level height will lead to systematic errors. 

The electrode positions should be properly positioned: The long electrode must reach the bottom of the culture plate; the short electrode is in the upper chamber. The electrodes must not touch the surface of the membrane to avoid puncturing the cell layer. 

The timing of measurement should be chosen correctly: Avoid measuring immediately after the fluid exchange. It is recommended to wait for 30 minutes to 1 hour to allow the cell state to stabilize. 

The cell confluence should be uniform: During the inoculation process, ensure that the cells are evenly distributed; if the growth is uneven, it will result in a lower and unstable TEER value. When transferring the culture plates, avoid shaking. 

Electrode maintenance should be done promptly: Before and after measurement, rinse the electrode with PBS to prevent salt crystals from blocking it. Regularly disinfect it with ethanol. 

The report should follow the MIRTA guidelines: When publishing, it is necessary to report the equipment model, electrode type, calibration method, membrane area, culture medium composition, and measurement temperature, etc.