Techniques to Assess Collagen Synthesis, Deposition, and Cross-Linking In Vitro

Synthesis, deposition, and cross-linking of collagen are hallmarks of fibroblast to myofibroblast differentiation. Standard methods for determining collagen from tissue samples are not directly applicable to cell culture conditions, where the overall synthesis and deposition of collagen is clearly unfavorable, mainly due to quantity limitations and dilution of required extracellular remodeling factors. In this chapter, we describe the methods we have established to analyze collagen production and deposition into the extracellular matrix by cultured myo/fibroblasts, as well as to determine lysyl oxidase (LOX) activity in cell supernatants as an index of the capacity of the cell to cross-link collagen in vitro.