Determination of collagen production

The collagen assay is based on binding of Sirius red dye to the triple helical collagen fibril. On days 7, 14 and 21 of culture, the cells were washed three times with PBS, and then 0.1% Sirius red in saturated picric acid was added (Sigma-Aldrich), and incubated for 60 min at room temperature. Then the plates were rinsed three times with 10 mM HCl. The presence of collagen was observed under an inverted microscope (Nikon Eclipse TE2000-u). The collagen-binding stain was finally eluted with 0.1 M NaOH and absorbance was read at 540 nm using the FLUOstar OPTIMA ELISA reader. Seven independent experiments were performed. The data are presented as means ± standard deviation. Student’s t-test was used for statistical analysis to compare the results. p < 0.05 was considered significant.