High-Throughout High-Sensitivity Lung FMT Assay

High-throughput, high-sensitivity FMT Assay

Accelerate your lead compound selection with predictive data from rapid screening of anti-fibrotic drug candidates.

Evaluate the effectiveness of therapeutic candidates targeting lung fibrosis

Determine compound efficacy using physiologically relevant assay conditions and high-throughput screening

Gather detailed insights with high-content, high-sensitivity imaging and advanced image analysis

High-throughput in vitro assessment of fibroblast-to-myofibroblast (FMT) transition using high-content imaging

The high-throughput, high-sensitivity FMT assay has been specifically designed to accurately and rapidly study a compound’s ability to prevent lung fibroblast activation and reduce the deposition of extracellular matrix proteins following stimulation with the well-established fibrotic mediator TGF-β1.

Our assay setup allows for advanced testing of small molecule drugs at multiple concentrations.

Using multiplexed detection, this FMT assay provides the accurate data needed to advance drug-discovery programs by determining changes in cell count, α-SMA expression, α-SMA strand perimeter and extracellular collagen I deposition.

The rapid, high-throughput fibroblast-to-myofibroblast transition (FMT) assay service leverages our state-of-the-art imaging suite capabilities to accurately evaluate the efficacy of anti-fibrotic compounds with fibrosis-related marker readouts. Automatic quantification and analysis ensures objectiveness in data quality.

FMT Assay service outputs

Cell count
Automated quantification and analysis of extracellular collagen I deposition
Automated quantification and analysis of α-SMA expression
Automated quantification and analysis of α-SMA strand perimeter

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Human lung fibroblasts stimulated with TGF-β1 to detect extracellular collagen I (pink), alpha-smooth muscle actin (α-SMA) (green) and cell nuclei (blue) as a measure of matrix production and fibroblast activation, respectively.

384-well format

4 parameter readout

High sensitivity

Assays

Lung fibroblast-to-myofibroblast transition (FMT) assay

Models

Primary human lung fibroblasts from healthy donor
Primary human lung fibroblasts from diseased Idiopathic Pulmonary Fibrosis patients

Timeline

4-6 weeks

Outsource your experiments to us

Why outsource your experiments to us

Take advantage of our knowhow and cutting-edge technology to obtain data which is reliable and predictive of in vivo activity.

Our in-house lung experts collaborate with you to design experiments focused on your hypothesis and perform them at our state-of-the-art facilities.

The fast and reliable high-throughput high-sensitivity FMT assay service coupled with our modern imaging suite capabilities provides study data that presents key insights into the ability of anti-fibrotic therapeutics to reduce lung fibroblast activation and matrix deposition in vivo.

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Service overview Close Open

Our FMT assay models the response of fibroblasts to TGF-β1 following a pre-incubation with test articles.

Assay Design
Cell Type	Primary lung fibroblasts
Species	Human
Assay Format	Cell count
	Quantification of extracellular collagen I deposition
	Quantification of α-SMA expression
	Quantification of α-SMA strand perimeter
Biological Variation	N = 3 validated healthy donors and N=2 validated diseased IPF patients lung fibroblasts
Technical Replicates	N = 6 technical replicates per condition
Assay Treatment Window	72 hours

Functionally validated assay Close Open

The assay is functionally validated showing
- Increased expression of α-SMA

α-SMA expression in response to dose dependent treatment with TGF-β1. Images were acquired using the ImageXpress Confocal HT.ai imaging system.

Increased α-SMA strand perimeter

TGF-β1 dose-dependently increases α-SMA strand perimeter indicating increased contractile potential of activated fibroblasts, while ALK5 inhibitor SB525334 shows a decreasing trend (A&B) Data for α-SMA strand perimeter from three healthy human lung fibroblast donors

Increased extracellular collagen I deposition

Extracellular collagen I deposition in response to dose dependent treatment with TGF-β1. Images were acquired using the ImageXpress Confocal HT.ai imaging system

Inhibition of TGF-β1 effect by exposure to ALK5 inhibitors SB431542 and SB525334 in response to pathophysiologically relevant TGF-β1 concentrations.

Controlled cell proliferation and optimal assay sensitivity Close Open

The addition of a macromolecular crowder (MMC) agent to the culture medium replicates a more in vivo like environment and promotes the deposition of secreted extracellular collagen I, thereby increasing the sensitivity of the signal.

Increased sensitivity. Addition of the macromolecular crowding (MMC) agent to the lung fibroblast culture medium increases the deposition of extracellular collagen I protein. (A) extracellular collagen I deposition. Images were acquired using the ImageXpress Confocal HT.ai imaging system (B) quantified expression levels of extracellular collagen I deposition.

Our FMT assay employs reduced serum concentration and optimized culture conditions that control cell proliferation even in the presence of varying doses of TGF-β1, thereby reducing the impact of changes in cell number on the expression levels of α-SMA and extracellular collagen I.

Controlled cell proliferation. Optimised assay culture conditions minimise the effects of TGF-β1 on nuclei count. Human lung fibroblasts from three healthy donors were stimulated with TGF-β1 and stained for cell nuclei.

Enhanced sensitivity and rapid screening Close Open

The HTHS FMT assay is highly sensitive in capturing minute changes in fibrotic marker expression that are critical in gauging anti-fibrotic drug efficacy. Laser-based α-SMA expression imaging and quantification yields a ‘steeper’ dose response curve with more area under the curve, delivering an overall higher sensitivity assay compared to LED-based imaging.

Higher sensitivity is achieved with laser-based imaging to acquire more subtle changes in fibrotic marker expression compared to LED-based imaging
Increased imaging speeds and reduced analysis time for rapid screening of large number of compounds

Quantified expression levels of α-SMA from ImageXpress Confocal HT.ai vs ImageXpress Pico. Data from three healthy human lung fibroblast donors (A, B & C).

Similar to quantification of α-SMA expression, the HTHS assay has been shown to generate more sensitive dose response curve for quantification of ECM proteins like extracellular collagen I deposition. This allows for more predictive dataset in screening anti-fibrotic compound screening.

Catalogue reference Close Open

Offering	Sku code	Format	Species	Readout
Offering	HTHS Lung FMT assay	LSFMT0000H	384-wells	Human	Cell count along with dose response curve for a-SMA expression, a-SMA strand perimeter and extracellular collagen I deposition

Images

α-SMA expression without(left) and with(right) TGF-β1 stimulation of primary HLFs acquired using ImageXpress Confocal HT.ai imaging system, Scale bar: 50µM

Extracellular collagen I deposition without (left) and with(right) following TGF-β1 stimulation of primary HLFs acquired using ImageXpress Confocal HT.ai imaging system, Scale bar: 50µM

Human lung fibroblasts stimulated with TGF-β1 and immunostained to detect extracellular collagen I (pink) and α-SMA (green) as a measure of matrix production and fibroblast activation respectively. Images captured using using ImageXpress Confocal HT.ai imaging system, Scale bar: 50µM.

Split view images with fluorescent staining of cell nuclei, α-SMA and extracellular collagen I (left) and respective segmentation analysis masks (right) for each stained marker under control (un-stimulated, top) and TGF-β1 stimulated conditions (bottom).

Increased resolution of laser-based acquisition. α-SMA staining following stimulation of human lung fibroblasts with TGF-β1 captured using LED-based imaging (left) vs laser-based imaging (right). Images taken at 20x magnification

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High-Throughout High-Sensitivity
Lung FMT Assay