Sircol assay for lung collagen quantitation.

Total soluble collagen content in right lungs was determined using the Sircol collagen-dye binding assay kit (Biocolor, Carrickfergus, N. Ireland). Briefly, whole right lung lobes from each mouse were homogenized in 1 mL of acetic acid solution (0.5 N with protease inhibitors) and incubated on a gentle shaker overnight at room temperature. The acid extracts were centrifuged (15,000g for 30 min) and aliquots of the supernatants (200 μl) containing acid-soluble collagens were mixed with 1 mL Sirius Red dye reagent for 30 min on a shaker (800 rpm). The pellets (bead-dye-collagen complex) acquired by centrifugation (10,000g for 10 min) were dissolved in 1 ml alkali reagent (0.5 M NaOH). Absorbance of alkali reagent in which collagen-dye complex was dissolved was measured at 550 nm. The amount of collagen was quantitated using a standard curve prepared from collagen standard.