The Cilia Beating Frequency (CBF) represents the average speed at which the epithelial cilia oscillate. It is expressed in Hz.
We measure CBF on our tissues with a high-speed camera (125fps), and an in-house developed software : Cilia-X.
This is a non-destructive endpoint.
The mucus moves at the surface of the epithelium. The Mucociliary Clearance (MCC) corresponds to its average travel speed, and is expressed in μm/s.
We measure MCC with a camera at 4 fps and a customized particle tracking software.
This endpoint is non-destructive but can be considered as a terminal endpoint.
Trans-Epithelial Electrical Resistance (TEER) is measured using the Epithelial Volt/Ohm Meter from Word Precision Instruments. TEER measures the tightness of the tissue; it reflects the integrity of the epithelium. Resistance is modulated by the presence of tight junctions and ion-channels.
TEER is a dynamic parameter that reflects the state of epithelia and is typically between 200 to 600 Ω.cm2. An increase of the TEER value reflects a blockage of the ion channel activities. A notable decrease of the TEER values (but > 100 Ω.cm2) could be observed in certain cases, reflecting an activation of the ion channels. Disruption of cellular junction or holes in the epithelia result in TEER values below 100 Ω.cm2.
When an epithelium is damaged, a decrease of TEER would be associated with an increase of LDH release or a decrease of the cells' viability.
This is a non-destructive endpoint.
Lactate dehydrogenase is a stable cytoplasmic enzyme that is rapidly released into the culture medium upon rupture of the plasma membrane. 100 μl basolateral medium collected at each time-point was incubated with the reaction mixture of the Cytotoxicity Detection KitPLUS (LDH) following the manufacturer’s instructions (Sigma, Roche 04 744 926 001). The amount of released LDH was then quantified by measuring the absorbance of each sample at 490 nm with a microplate reader. To determine the percentage of cytotoxicity, the following equation was used (A = absorbance values):
Cytotoxicity (%) = (A (exp value)-A (low control)/A (high control)-A (low control))*100.
The high control value was obtained by 10 % Triton X-100 apical treatment (24 hours). Triton X-100 causes a massive LDH release and corresponds to 100 % cytotoxicity.
This is a non-destructive endpoint.
Cytokines, chemokines, metalloproteinases and antimicrobial peptides are secreted constitutively or upon challenge in the basolateral or apical side of the airway epithelium.
These soluble factors are quantified using ELISA. Typical markers include among others : IL-8, IL-6, IP-10, IL-1 beta, RANTES, GRO-alpha, TGF-beta, GM-CSF, Interferon-gamma, MMP-9, beta-defensin, cathelicidin...
This is a non-destructive endpoint.
Pseudo-stratified architecture as well as cellular organization are visualized using histological analysis / tissue cross sections. The following stainings can be used :
This endpoint is destructive and should be considered as a terminal endpoint.
Mucin secretion is quantified using an ELLA (Enzyme-linked Lectin Assay) in-house protocol detecting the carbohydrates groups of the collected mucus.
This is a non-destructive endpoint.
Since each epithelium is made of 500'000 cells, large amounts of DNA / RNA can be isolated, allowing quantification of genes by RT-qPCR
This endpoint is destructive and should be considered as a terminal endpoint.
Proteins can be detected using western blot technique.
This endpoint is destructive and should be considered as a terminal endpoint.