2.5. TM cell collagen secretion

Collagen secreted into the media by the TM cultures was analyzed using the Sircol Assay Kit (Biocolor, Westbury) according to manufacturer’s instructions First, the TM cells were seeded into a 6-well plate at 2.0×10⁵ cells/well and incubated for 4-hours. Then, the media was replaced with conditioned media either containing Dex (100 nM), Dex with compounds, DMSO (vehicle), and incubated at 37°C for 48-hours. After incubation, 1 ml of media was collected from each tube and were mixed with 200μL of Collagen Isolation and Concentration Reagent from the Sircol Assay Kit and incubated overnight at 4 °C. The following day, the samples were centrifuged at 12,000 rpm for 10 minutes and the supernatants were discarded. Then, 1mL of Sircol dye reagent (Sircol Assay Kit) that binds to collagen was added to each tube and gently mixed by inverting. The tubes were incubated for 30 minutes at room temperature and were then centrifuged at 12,000 rpm for 10 minutes. After, 750 μL/tube of ice cold Acid-Salt Wash (from the Sircol Assay Kit) was gently layered over the collagen-dye pellet, mixed and centrifuged at 12,000 rpm for 10 minutes to remove any unbound dye. The supernatant was disregarded and 250 μL/tube of the alkali reagent was added to the collagen-bound dye. The pellet formed was vortexed until completely dissolved back into solution and 200 μL aliquots of each sample was loaded on a 96-well plate. Using the FilterMax F5 microplate reader (Molecular Devices, Sunnyvale, CA), absorbance was measured at 555nm.