Fig. 3

Overview of the ‘scar-in-a-jar’ assay and HCS algorithm to analyse nuclear integrity and collagen deposition. a Overview of the ‘scar-in-a-jar’ assay from seeding cells on the first day to adding compounds, TGF-β₁ stimulation on the second day (T₀), cell fixation and performing immunocytochemistry (ICC) to quantify cell number and collagen deposition at 72 h post-stimulation (T₇₂). b Representative images of Hoechst-stained nuclei (350 nm/460 nm) and AlexaFluor488 (490 nm/525 nm)-collagen type I immunoreactivity acquired using the CellInsight at 10x magnification. A computational algorithm identified viable Hoechst-stained nuclei (blue) and excluded nuclei (yellow). Green indicates collagen mask applied to fluorescence above the background signal. Scale bars represent 200 μm. c & d Using a modified version of the ‘Cell Health Profiling v4’ algorithm, total fluorescent intensity (c) and mean fluorescent intensity (MFI; (d)) were quantified. Data points represent mean ± SEM (n = 9 independent experiments). ***P ≤ 0.001 as determined by Mann-Whitney statistical analysis