Barrier Function

Barrier application
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In the body, cellular barriers line many surfaces to protect the tissue within and control the passage of material in and out. These barriers are primarily composed of endothelial and epithelial cells. Biological events such as inflammation, infection, cancer metastases, leukocyte migration and many “normal” cues (e.g. GPCR) can alter the permeability of the barrier.

Axion BioSystems' Maestro Z, TrayZ, ZHT, Pro, and Edge systems offer impedance-based cell analysis for real-time, continuous, label-free monitoring of your cells. Continuous data reveals the full time course of barrier disruption for a more complete picture without the time- and cost-intensive process of repeating multiple endpoint assays. 

Track barrier integrity in real time
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Transepithelial electrical resistance (TEER) is commonly used to measure barrier integrity.

 

 

The Maestro impedance platform can measure this resistance and expands on it with barrier index. By simultaneously measuring at different frequencies, the Maestro impedance platform calculates barrier index by normalizing TEER values for confluence, providing a sensitive and less variable measure to small, transient disruptions in barrier function. Barrier permeability can be altered by many common drugs and signaling molecules. Here, TEER was monitored continuously for Calu-3 cells in a 96-well CytoView-Z plate on the Maestro Z system. After reaching confluence, cells were dosed with cytochalasin D and ethanol. Cytochalasin D inhibits actin polymerization to increase tight junction permeability, as reflected by the rapid decrease in resistance. Ethanol acts more slowly on ZO-1 and claudin-1, two key tight junction proteins. The rapid disruption, as well as the distinct dynamics, are captured by TEER measurements on the Maestro Z.

TEER can be measured continuously.
Monolayers cultured for 14 days and treated with Ethanol which significantly reduced TEER

(A) Barrier permeability can be measured continuously from proliferation to confluence. (B) Calu-3 monolayers were cultured for 14 days in the CytoView-Z 96-well plate and then treated with cytochalasin D and ethanol, both of which significantly reduced the TEER.

Evaluate barrier function in disease-in-a-dish model
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Barrier properties serve critical functions throughout the human body and may be disrupted by diseases, such as cystic fibrosis. Calu-3 is an immortalized cell line that produces tight junctions and expresses functional cystic fibrosis transmembrane conductance regulator (CFTR), which allows ion transport across the cell membrane upon β-adrenergic stimulation [Shen et al., 1994]. TEER measurements capture the function of CFTR, which opens conducting ion channels to facilitate mucus secretion, a process disrupted in cystic fibrosis.

Addition of isproterenol significantly reduced TEER
Measurements cconfirming CFTR as the mechanism for action for the change in TEER

(C) Addition of isoproterenol (teal, 100 nM) and forskolin (orange, 10 µM) significantly reduced TEER of the Calu-3 cells. CFTR(inh)-172 was then added to the plate at 30 and 300 µM causing a rapid dose-dependent reversal of the isoproterenol and forskolin response. (D) 5 minutes after CFTR(inh)-172 addition isoproterenol and forskolin responses are inhibited in a dose-dependent manner.

Assay Steps
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Barrier protocol assay steps

Getting started with Maestro Z, ZHT, Pro, and Edge couldn't be easier. Culture your cells in an Axion multiwell CytoView-Z plate (Hour 0). Load this plate into the Maestro system and allow the environmental chamber to automatically equilibrate. Observe cells adhering to the plate and proliferating as changes in the recorded impedance signal (Hour 0 to 24-72). Add test compounds as required. Track changes in barrier integrity in the CytoView-Z plate label-free and in real-time with the Impedance Module software.