PURPOSE: Idiopathic pulmonary fibrosis (IPF) is the most common form of idiopathic interstitial pneumonia. Numerous inflammatory and pro-fibrotic cytokines may contribute to the development of this disease. Among these, transforming growth factor beta (TGF-?) plays a significant role in collagen accumulation by inducing expression and suppressing degradation of collagens. Undesirable accumulation of collagens results in the development of numerous diseases, including pulmonary fibrosis. Previous techniques, which were time-consuming and expensive, were based on the evaluation of radio-labeled proline, quantification of hydroxyproline using high performance liquid chromatography, or precipitation of collagen using Sirius Red.
METHODS: We describe a new plate-based high throughput screening of collagen synthesis using Sirius Red as a dyeing reagent. Human lung fibroblasts (HFL-1) were grown to post confluency; ascorbic acid (20 ?g/mL), proline 10 (?Mol), and TGF-? (0.25, 0.5, and 1.0 ng/mL) were added for 72 hours. Medium was removed, and cells were fixed with 0.5% Glutaraldehyde for 30 minutes at room temperature. Glutaraldehyde was removed, cells were washed with distilled water (ddH2O), and the first 24 wells, which were used to determine collagen synthesis, were treated with 0.5 Molar acetic acid for 30 minutes. The second 24 wells were stained with 0.1% crystal violet for viability. After washing with ddH2O, 250 ?l of Sirius Red was added to acetic acid-treated cells for 2 hours, removed, and wells were washed with ddH2O.
RESULTS: Absorbed Sirius Red and crystal violet were eluted with 300 ?l of alkaline solution and 0.1 Molar citric acid, and absorbances were determined at 540 and 600 nm, respectively. The extent of collagen accumulation was determined by normalizing the levels of induced collagen to cell density. Results were validated by quantifying the suppression of TGF-?-induced collagen synthesis by pirfenidone and interferon gamma-1b and comparing the results to those obtained using the conventional Sirius Red procedure.
CONCLUSION: The high throughput screening collagen assay is a reliable, efficient technique for evaluating collagen metabolism.
CLINICAL IMPLICATIONS: Our method provides an opportunity for high throughput screening of collagen induction/repression by biological/nonbiological factors.
DISCLOSURE: Osman Ozes, University grant monies No; Grant monies (from sources other than industry) No; Grant monies (from industry related sources) No; Shareholder O. Ozes, L. Blatt: InterMune shareholders; Employee O. Ozes, L. Blatt: InterMune employees; Fiduciary position (of any organization, association, society, etc, other than ACCP No; Consultant fee, speaker bureau, advisory committee, etc. No; Other No; Product/procedure/technique that is considered research and is NOT yet approved for any purpose, All.