Tissue fibrosis is characterized by chronic fibroblast activation and consequently
excessive accumulation of collagen-rich extracellular matrix. In vitro microplate-based
assays are essential to investigate the underlying mechanism and the effect of antifibrotic
drugs. In this study, in the absence of a gold-standard method, we optimized a simple,
cost-effective, Sirius Red-based colorimetric measurement to determine the collagen
production of fibroblasts grown on 96-well tissue culture plates. Based on our findings,
the use of a serum-free medium is recommended to avoid aspecific signals, while ascorbate
supplementation increases the collagen production of fibroblasts. The cell-associated
collagens can be quantified by Sirius Red staining in acidic conditions followed by
alkaline elution. Immature collagens can be precipitated from the culture medium by
acidic Sirius Red solution, and after subsequent centrifugation and washing steps,
their amount can be also measured. Increased attention has been paid to optimizing
the assay procedure, including incubation time, temperature, and solution concentrations.
The resulting assay shows high linearity and sensitivity and could serve as a useful
tool in fibrosis-related basic research as well as in preclinical drug screening.
