TEER troubleshooting

How to Troubleshoot TEER Measurement Problems

By Subhra Nag, PhD

Transepithelial/transendothelial electrical resistance (TEER) measurement is one of the most used practices to evaluate cellular health, such as cellular confluence, barrier integrity, or barrier function of cellular monolayers grown onto multiwells. The TEER measurement using WPI’s Epithelial Voltohmmeter (EVOM) is considered the gold standard because of its reliable measurements and numerous literature citations using various cell types. The EVOM™ Manual and EVOM™ Auto along with different choices of electrodes (STX4, STX HTS high throughput screening, EndOhm chambers, and multielectrode array for EVOM™ Auto) allow researchers to perform and analyze cell samples in 6, 12, and 24 removable inserts and 24 and 96 HTS multiwell plate formats. The major challenges researchers may encounter while performing studies to capture TEER measurement include:

  • Unstable read outs
  • Out of range values
  • Inconsistent measurements among sample replicates or batches.

Consider the following factors to overcome any TEER measurement problems and obtain accurate and reliable measurements:


Presence of Conductive Liquid in Samples

To get stable measurements, the electrode tips (active sensing regions) need to stay immersed in a conductive liquid such as, 1XPBS buffer, cell culture media, etc. Deionized water will not work. For the EVOM’s applied current to flow through the samples, the solution needs to have ions (e.g., chloride ions) present. Ionic concentrations and measured electrical resistance values maintain an inverse relationship. With increasing ionic strengths or concentrations, the resistance value should decrease inversely. To achieve stable and consistent measurements and to make meaningful comparisons, use the same media or buffer solutions in all samples.


Adequate Liquid Volumes

The volumes of the liquid on top (apical) and bottom (basolateral) of the membrane of the multiwell or cell culture insert need to be adequate so that the electrode tips can stay fully immersed in the conductive solution. Lack of adequate volumes of liquid can result in unstable readings or out of range values, since the electrode cannot consistently pass the electrical current across the samples. If the membrane of the cell culture insert is ruptured, the apical liquid will easily move towards the basolateral side. These samples should be omitted from studies since the electrical current can move through the sample liquid leaking path and would provide inaccurate estimation of electrical resistance. Use consistent volumes of liquids to get consistent read outs. 


Select an Electrode Matching Insert/Plate Type

WPI offers a wide variety of electrodes matching the dimension and geometry of commonly used cell culture inserts (e.g., Corning, Millipore, MatTek, Greiner) with different sizes (6, 12, 24, and 96 multiwells). WPI recommends using a matching electrode with the cell culture insert or muliwell type for the best results. For example, for Corning 3460 12 well inserts, the ENDOHM-12 needs to be used (Part number ENDOHM-12G for EVOM2 or EVM-EL-03-01-02 for EVOM™Manual). The data gathered with a mismatched electrode may be significantly inaccurate. 


Position of the Electrode Over Samples

The position of the electrode (including its depth into the samples) can affect raw resistance readings. Consistent electrode placement provides a fixed or consistent path for applied electric current flow through samples and eliminates or minimizes variations of electrical resistance readings. WPI’s advanced electrodes, such ENDOHM or STX HTS, when properly used with matching multiwells, minimize this reading variability due to the ability of consistent electrode positioning. Using EVOM™ Auto, its automated robotic arm operated movement also eliminates this effect when same electrode array/plate profile setting is being consistently used. 


Use of a Blank and Blank Subtraction

The “blank sample” refers to a cell culture insert or multiwell without cells which also conforms to the same experimental conditions (including same liquid type and volumes). Subtracting the blank resistance value from sample resistance and then calculating TEER values helps to minimize the effects of variability parameters, such as electrode positioning, sample liquid type and volume. Both EVOM™ Manual and EVOM™ Auto systems allow you to save the blank values and automatically subtract them from sample resistance values. 


Measure Samples at Stable Temperature

WPI recommends taking the plates out of the incubator to acclimate the samples at room temperature inside the cell culture laminar hood for 20 minutes before taking measurement. When a plate is taken right out of a 37°C incubator and measured, large sample-to-sample variations may be seen since sample 1 may be at 37°C, whereas sample 12 of the same plate may be at 32°C. Similarly, room temperature acclimated media/solution is to be added in case any solution is added before measurement. The temperature is known to affect the tight junction permeability, and hence can affect the TEER readings. Therefore, measurements need to be taken at a stable temperature to obtain stable readings. 


Cleaning and Maintenance of the Electrode

The regular/daily electrode cleaning and maintenance is critical for functional longevity of the electrode. The electrodes tend to accumulate salt and protein deposits from the media or buffer if not immediately cleaned after use or at the end of the day. This deposition may block the active sensing region of the electrode and can result in low resistance or unstable readings. WPI recommends regular cleaning of the electrode at the end of the day after use to ensure the electrode sensing region does not form deposits. 


The electrode tips are normally washed with ethanol or isopropanol, followed by a rinse with DI water. The electrode tips need to air dry, and the electrode should be stored in a dry condition. Periodic cleaning of electrode tips using proteolytic detergents such as Enzol or Alconox, is recommended to prevent protein deposits. The remaining portion of the electrode other than the tip with the active sensing area, cannot be immersed in any liquid, and can be wiped with a paper towel sprayed with alcohol. Periodic chloride the electrode tips or sensing region by keeping the electrode tips immersed in 3-6% sodium hypochlorite (bleach) for 10 minutes followed by a rinse with DI water. This is recommended to replenish the silver chloride for proper electrode functionality and stable readings. Refer to the specific electrode’s cleaning and maintenance section of the instruction manual for additional details.
 
Following the suggestions above for your TEER measurement experiments, you can expect to overcome TEER measurement problems and achieve stable and reproducible results. If you have an questions, just give us a call at (866) 606-1974  or email us at [email protected].

 

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