Sircol assay

To investigate the effect of mechanical loading on the secreted ECM proteins, Dimethylmethylene Blue Assay (DMMB) assay and Sircol assay (Biocolor, UK) were used to quantify the soluble sGAG and collagens, respectively, in the cell culture media. At the day of characterization, the cell culture media were collected. A portion of the media was mixed with either DMMB dye or Sircol dye and incubated with moderate agitation at room temperature for 30 min. Upon incubation, the solution was centrifuged to form a pellet of sGAG or collagens that bound to the respective dye. The pellet was washed in ice-cold acid-salt solution, centrifuged and resuspended in 10% SDS for DMMB assay or Alkali solution for Sircol assay. For DMMB assay, absorbance at 656 wavelength while for Sircol assay absorbance at 555 wavelength was quantified using a microplate reader (SOFTmax Pro, USA). Absorbance was converted to sGAG concentration or Collagen concentration using a calibration curve obtained using different concentrations of chondroitin sulfate (Sigma-Aldrich, USA) or Type-I collagen (Corning, USA), respectively. The sGAG or collagen concentrations were normalized using the total protein in the media quantified using UV 280 wavelength absorbance and converted to protein concentration through a calibration curve obtained by diluting a stock solution of Bovine Serum Albumin (BSA, Sigma-Aldrich, USA).

For lung tissue collagen assay, 0.05 g lung tissue was homogenized in 0.5 M acetic acid (1 mL) containing 7.5 mg pepsin, and rotated for 24 h at 4 °C. Samples were briefly centrifuged to pellet debris, and 100 µL of each supernatant was assayed for collagen by Sircol assay as described by the manufacturer (BioColor Ltd., Carrickfergus Belfast, UK). For measuring soluble collagen in BAL fluid, aliquots of 25 µL were subjected to Sircol assay. Collagen concentration was determined by absorbance at 540 nm in a spectrophotometer and titration according to standard curves generated for lung tissue and BAL fluid.