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.

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

*Vehicle and untreated controls also included.