Application NoteTopic:
TEER (TransepithelialElectrical Resistance)

Monitoring cell – attachment and tightjunctions in the same assay

Cells:MDCK-II epithelial cells
Tools:AtlaZ

Nanion Technologies GmbH, Munich.
University of Regensburg, Regensburg.

Summary
The numerous different cell types in the human body are

examination with a minimum disturbance of the cells or tissues

greatly specialized and often require a conjunctive action

being investigated (5).

of a population of cells, for example in tissues. Cells areinterconnected via cell junctions, multiprotein complexes

Here, we used MDCK-II cells, which are a well establishedfound in the cell membrane of animal cells, and such cellepithelial cell line used to study tight junctions (6). After

junctions allow for a mechanical, chemical or electricalcompletion of cell adhesion and formation of a confluent

transmission of signals. These junctions can be subdivided intomonolayer, the barrier forming tight junctions between(I) tight junctions, (II) anchoring junctions or (III) gap junctions.

adjacent cells are developed (Figure 1). The formation of tight

Defects in cell–cell junctions give rise to a wide range of tissue

junctions requires fully established cell-surface contacts.

abnormalities that disrupt homeostasis and are common in
genetic abnormalities and cancers (1).

The platform used here, AtlaZ, is a quantitative live-cell analysis

The so-called tight junctions form the barrier in endothelial andsystem and allows for cellular research on cell adhesion

epithelial cells. Classical transepithelial electrical resistanceand proliferation, cytotoxicity, GPCR, morphology andmeasurements are performed using microelectrodes, wherebarrier function, label-free and in real-time. Recordings cantrans- and para-cellular conductivities can be calculated (2).

be performed in up to six 96-well plates simultaneously or

Here the cells are grown on a porous filter membrane which isindependently.

placed between two fluid compartments. Flux of solutes fromone compartment to the other must pass the interfacial cell

The impedance of planar gold-film electrodes that arelayer then, and this is determined by the functional porperties

used as growth substrate for adherent cells reveals changesof the tight junctions.

in electrode coverage (Figure 1) or cell behavior. Realtime impedance data provide insights in cell morphology,

Electrical impedance spectroscopy (EIS) (3, 4) as themethodology behind the AtlaZ system, is based on thea system while it is excited with a low ampltidue alternating
current or voltage. One important property of EIS is its

1

Adherent epithelial cellGold electrodenoninvasiveness, as the technique relies entirely on low-Cell Signal

opposition of current flow, on measuring the impedance of

amplitude currents and voltages that sensure damage-free

Proliferation
Time (h)Proliferation & developmentof a confluent monolayer

Cell Signal

2

Confluent monolayer (plateau phase)Time (h)forming cells proliferate after seeding. Once aconfluency of cells is reached, cells start formingtight junctions which are sealing the paracellulargap. The TEER signal then is recorded in parallel tocell adherence.

TEER

3

Confluent cells develop tight junctions
Cell Signal

Figure 1: Workflow for TEER assay. Tight junction

Confluent cells developTime (h)proliferation, lateral migration or cytotoxicity even over

It is well known that the formation of cell-matrix (surface)prolonged periods of time.

junctions is dependent on the presence of either Ca2+ or Mg2+(or Mn2+), whereas tight junction formation is known to depend

A crucial advantage over standard assays, e.g. filter basedon the presence of Ca2+, which cannot be substituted by amethods using labels and either an optical or radiometricdifferent cation. To prove this, and, furthermore to show that

detection technique, is the continuity of cell monitoring.

the increase in TEER in Figure 2B is due to the development oftight junctions, we next cultured cell populations in either Ca2+

Unique culture plates with integrated electrodes as used inor Mg2+ containing buffer (Figure 3). In the presence of only

the AtlaZ system enable long-term measurements over several

Mg2+, the tight junctions could not develop, whereas in theweeks. Thus real-time data on cell adhesion, proliferation andpresence of Ca2+ the TEER due to tight junctions increased

barrier integrity can be aquired.

until a plateau phase was reached and then the junctions
remained stable (Figure 3).

Results

Taken together, the results show that it is possible to monitor
the formation of cell – surface and cell-cell contacts in one

Here MDCK-II cells were used which are a well establishedand the same cell population (well) by utilizing non-invasive

epithelial cell line developing tight junctions. Cells were

electrical impedance spectroscopy measurements in real

seeded on non-coated AtlaZ sensor plates at time zerotime.

(Figure 2B). The initial cell density was sufficiently high sothat a confluent monolayer could form within 6 - 7 hours. A

In summary, AtlaZ allows for label-free and real-time

plateau phase of the Cell Signal represents full coverage ofcellular research on cell adhesion, proliferation and TEER

the surface with adherent cells. From the timepoint at whichmeasurements. The AtlaZ system provides a versatile tool

the cell adhesion was complete (dashed line in Figure 2B), thefor in vitro cell monitoring addressing the demands fortransepithelial electrical resistance (TEER) began to increase

versatility, physiological relevance and throughput.

considerably for the next hours, indicating the formation ofbarrier forming tight junctions between adjacent cells.
In general, the Cell Signal recorded with AtlaZ is dominatedMethods

by the paracellular cell layer resistance or capacitive currents
across the cell membranes – depending on the readout
frequency (Figure 4). Figure 2A shows a schematic depicting
low and high frequency impedance recordings for para- andtrans-cellular resistance, representing cell coverage andstrength of barrier formation, respectively.

AtlaZ platform
The AtlaZ platform (Nanion Technologies) provides
quantitative live-cell analytics by measuring the impedance(Ohm, W) of adherent cells as grown on 96-well plates withembedded planar gold-film electrodes (Nanion Technologies).

The data in Figure 2B show that the formation of tight junctions
requires fully established cell-surface contacts.

A crucial advantage over standard assays is the continuity ofcell monitoring. Continuous measurements reveal the kinetics
of cell behavior and allow an in-depth mechanistic
understanding without the need for time- and labor-intensiveAB

endpoint assays. For example, one of the crucial advantages

Low frequency(TEER)High frequency(cell attachment)

cell layer
extracellular matrix
gold electrodeof data derived from the AtlaZ platform is the possibility toanalyse dose-responses at any time during the experiment.
Advanced information content is obtained by using multi-cell attachment
TEER

formation ofcell-surface
contact

formation ofbarrier formingtight junctions
betweenadjacent cells

Figure 2: A Low and high frequency impedance
recordings for cell-cell contact or cell attachment
measurements. B Cell coverage, measured at 50
kHz increases over time (grey) and TEER, measured
at 1 kHz (black) reveals barrier formation.

Figure 3: In the presence of only Mg2+, thetight junctions cannot develop, whereas in thepresence of Ca2+ the cell-cell contacts via tightjunctions build an increasing TEER. The resistanceincreases and then reaches a plateau phase andremains stable after approx. 2 h after cell seeding.

frequency impedance readouts, which is possible with theAtlaZ Control software. With impedance readouts at 1000
different frequencies (here a spectrum of 0.1 kHz – 100 kHz,see Figure 4) it is possible to further dissect physiologicalresponse into deeper levels, and thus zooming in on changes
in membrane topography, cell-cell or cell-matrix junctions isachievable. G-protein-coupled receptor (GPCRs) activation is
one example here.

References

Workflow of AtlaZ TEER assay

1. Garcia MA, Nelson WJ, Chavez N. Cell-Cell Junctions Organize

Target cells are seeded in AtlaZ sensor plates and they adhereon the surface with embedded gold electrodes (Figure 1).
Each well of the 96-well plate contains 1 center gold electrodewith 0.6 mm in diameter and one reference electrode. Afterthe target cells being adhered, the population starts toproliferate. When the cells reach confluency, cells start formingtight junctions which are sealing the paracellular gap. TheTEER signal then is recorded in parallel (Figure 4) to the cellattachment, proliferation, cell death or morphology changes
in the presence of drugs or stimuli of interest.

Structural and Signaling Networks. Cold Spring Harb Perspect
Biol. 2018 Apr 2;10(4):a029181. doi: 10.1101/cshperspect.
a029181. PMID: 28600395; PMCID: PMC5773398.
2. Gitter, A.H., Bertog, M., Schulzke, J. and Fromm, M. (1997)
Pfügers Archiv. European Journal of Physiology, 434, 830.
3. Reiß, B. and Wegener, J. (2015) Impedance Analysis ofdifferent cell monolayers grown on gold-film electrodes.
IEEE Eng. Med. Biol. 7079-7082. org/10.1002/9780470015902.
a0025710
4. Lukic, S.; Wegener, J. (2015) Impedimetric monitoring of cell-

Acknowledgement
MDCK-II cells were kindly provided by Prof. Dr. Joachim
Wegener, University of Regensburg.

based assays. Encyclopedia of Life Sciences, Wiley & Co.
https://doi.
5. Wegener, J., Impedance Analysis of Cell Junctions (2019),
https://doi.org/10.1002/9783527628155.nanotech073
6. Dukes JD, Whitley P, Chalmers AD. The MDCK variety pack:choosing the right strain. BMC Cell Biol. 2011 Oct 7;12:43.
doi: 10.1186/1471-2121-12-43. PMID: 21982418; PMCID:
PMC3209442.

Figure 4: Electrical impedance spectroscopy.

Cell
Monitoring

AtlaZ allows to detect impedance signals at 1000
different frequencies, ranging from 0.1 kHz – 100

TEER

kHz. TEER signals representing barrier integrity can(TransepithelialElectricalResistance)

Cytolysis

be recorded in parallel to cell adherance whenanalyzing the data output at 1 kHz or 50 kHz,respectively. Predefined recording modes can bechosen for ease-of-use.

tightjunctions

barrier
integrity

0.1

1cell coverage andstrenght of barrier

wound
healing

proliferation /cell density

Recording frequency (kHz)TEER Mode

1025

Cell Monitoring Mode

50100

Cytolisis Mode

Key findings
1. Our results demonstrate that cell-cell and cellsurface contacts can be recorded in parallel.
2. AtlaZ allows for cellular research on cell adhesion,proliferation and cytotoxicity, label-free and in realtime.
3. Recordings can be performed in up to six 96-wellplates simultaneously or independently.

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