4.7. Red Sirius Staining for Total Collagen Quantification

The Red Sirius staining procedure allows quantifying total collagen. Picrosirius red, a strong linear anionic dye comprising six sulfonate groups, associates along cationic collagen fibers.

After incubation, the culture medium was removed, and cells were rinsed twice with PBS 1X (Fisher Scientific, Illkirch, France). Then, 1 mL of Bouin solution (Sigma-Aldrich, Saint Quentin Fallavier, France) was added in each well, and cells were stained for 1 h at room temperature. Then, cells were rinsed twice with distilled water and dried under the hood. Cells were stained with 1 mL of Red Sirius solution (Sirius Red 80 in aqueous saturated picric acid solution, Sigma-Aldrich, Saint Quentin Fallavier, France) for 1 h. After staining, solution was removed, and cells were rinsed successively with distilled water and HCl 0.01N to remove unbound dye. Bound dye was solubilized with NaOH 0.1N for 30 min. Colored solution was transferred to a new 96-well microplates, and an absorbance read was performed at 550 nm (Varioskan Lux, Thermo Fisher Scientific, Vantaa, Finland).