Determination of Lung Hydroxyproline Content

Lung collagen at the end of 21 days was determined by estimating the amount of hydroxyproline present in the tissue sample using the Hydroxyproline Assay Kit (Sigma-Aldrich, Saint Louis, MO, United States) as per the manufacturer’s instructions. Briefly, lungs were cleared off the extraneous material and washed with PBS. 10mg of tissue was homogenized in 100 μl of ice cold Milli-Q water and transferred to a pressure-tight glass vial. Hundred microliter of 12N HCl was added to the vial, capped tightly, and hydrolyzed at 120°C for 3 h. The hydrolysate was then separated from the particulate matter by centrifugation. Twenty five microliter of sample was transferred to a 96-well plate and dried in a 60°C oven. Hundred microliter Chloramine-T solution was added to the wells and incubated at room temperature for 5 min, followed by incubation with 100 μl Ehrlich’s Solution for 90 min at 60°C. Absorbance was measured at 550 nm and hydroxyproline concentrations in the sample was calculated from the standard curve generated using known concentrations of trans-4-hydroxyl-L-proline (Sigma H5534). Results were expressed as micrograms of hydroxyproline per ml sample.