Lung Fibrosis Assay Services
Commonly-used fibrosis assays are often either too low throughput or are poorly predictive of downstream success. Our R2G Lung Fibrosis Assay Service can help you find better candidates in early drug discovery.
Key Features & Benefits
Control fibrosis induction in 2D model. Cell culture protocols limit stress-fiber formation.
Pathologically relevant readout. ɑ-SMA in stress fibers are measured.
High-content analysis. Cell morphologies and other phenotypes are measured in addition to ɑ-SMA.
Uses a physiologically relevant model. Primary normal human lung fibroblasts (NHLF) cells.
R2G assay services are a starting point. Transition to more complex, bespoke assay services with the same service provider.
Assays Designed with Your Goals in Mind
Fibrosis is a pathological state that arises from abnormal tissue repair, whereby connective tissue replaces healthy tissue to an extent that disrupts normal functioning of the organ. It is characterized by the accumulation of proteins in the extracellular matrix, which results from prolonged activation of myofibroblasts following various types of severe or repetitive tissue damage. Prolonged myofibroblast activation induces the formation of alpha smooth muscle actin (α-SMA)-containing stress fibers, a common marker in fibrosis assays including our R2G Lung Fibrosis Assay Service.
Most current fibrosis assays are not well suited for early stages of drug discovery. Simpler models of fibrosis often fail because standard cell-culture procedures tend to induce fibrosis-like phenotypes, making it difficult to distinguish between healthy and diseased states. In contrast, while 3D models of fibrosis can distinguish the two phenotypes, they are not amenable to high throughput screening, which limits the potential and efficiency of drug-candidate identification.
Our Ready-2-Go (R2G) Lung Fibrosis Assay Service uses primary, normal human fibroblasts (NHLF) cells cultured using a protocol that limits basal induction of fibrosis (which is often observed with routine, cell-culture procedures) to generate a 2D model of fibrosis. This not only allows for enhanced control of fibrotic induction, but also affords a format amenable to higher-throughput therapeutics profiling.
How It Works
Primary normal human lung fibroblasts (NHLF) are cultured using a specialized protocol that limits induction of fibrosis prior to running the experimental procedure. Cells are seeded into 384-well plates and compounds are added to the cells for 30 minutes before inducing fibrosis with TFG-β. After 48 hours and 72 hours of TGF-β stimulation, cells are fixed and stained (see below) for imaging and analysis.
The representative images of NHLF cells shown above were taken following 48-hour exposure to either vehicle (PBS) (left), TGF-β (middle), or TGF-β + inhibitor ALK5i (a.k.a. Galunisertib) (right). The images show a visible increase in the levels of actin filaments and α-SMA-containing fibers increased with TGF-β treatment, a phenotype not observed when cells were pre-treated with the inhibitor ALK5i. This observation was quantified by measuring the total phalloidin intensity at different concentrations of TGF-β, with and without ALK5i inhibitor present, and is shown on the graph on the right.
Assay Service Details
|Ready-2-Go Lung Fibrosis||Bespoke Assay Services|
|Cells||NHLF (Lonza, CC-2512)||If you would like to expand the service offering beyond R2G shown on left, please contact us.|
|Markers||Hoechst (nuclei), Phalloidin (F-actin), and α-SMA antibody|
|Dosing||6 doses of your test article|
|Negative Controls*||1D-11; Alk5i (Galunisertib)|
|Replicates||Triplicates (at a minimum)|
|Assay Readouts||Phalloidin intensity, nuclear count, α-SMA intensity in phalloidin-positive fibers, Cell Morphology (cell area, length-to-width ratio)|
*Vehicle and untreated controls also included.