Quantitative Analysis of Activated Fibroblasts

The Operetta assay was performed as previously described (Schafer et al., 2017 (link)) with minor modifications: HCFs were seeded in 96-well black CellCarrier plates (PerkinElmer) at a density of 5 × 10³ cells per well. Following simulations, cells were fixed in 4% PFA (Thermo Fisher), permeabilized with 0.1% Triton X-100 (Sigma) and non-specific sites were blocked with 0.5% BSA and 0.1% Tween-20 in PBS. Cells were incubated overnight (4°C) with primary antibodies (1:500), followed by incubation with the appropriate Alexa Fluor 488 secondary antibodies (1:1,000). Cells were counterstained with 1 μg/ml DAPI (D1306, Thermo Fisher) in blocking solution. Each condition was imaged from duplicated wells and a minimum of seven fields/well using Operetta high-content imaging system 1,483 (PerkinElmer). Cells expressing ACTA2 were quantified using Harmony v3.5.2 (PerkinElmer) and the percentage of activated fibroblasts/total cell number (⍺-SMA^+ve) was determined for each field. The measurement of fluorescence intensity per area (normalized to the number of cells) of Collagen I was performed with Columbus 2.9 (PerkinElmer).