Griener

TED orbital fibroblasts were plated in black 96-well plates (Griener, Sigma-Aldrich) at 5 x 10³ cells/well with 200 μl of culture medium. Vehicle (DMSO) or PPIs were added every 24 hours starting with 24 hours before addition of TGFβ (1 ng/ml) for 96 hours to individual wells run in triplicate. Puromycin (5 μg/ml) was used as a positive control for cytotoxicity and was added once. The Alamar blue reagent (Invitrogen, Carlsbad, CA) was added to each well 24 hours after the addition of TGFβ. Fluorescence of the oxidized Alamar reagent was measured after 48 hours (72 hours of incubation with TGFβ and 96 hours with PPIs) (excitation 470 nm, emission 480 nm).

Ad-MSCs were plated in black 24-well plates (Griener; Sigma-Aldrich, St Louis, Mo) containing either no matrix or dressing C or A with 800 μL of culture medium. Vehicle (PBS), TGFβ (5 ng/mL), or IL-1β (100 pg/mL) was added, cells were incubated for 24 hours, and then Alamar blue reagent (Invitrogen) was added to each well as directed by the manufacturer's instructions. Fluorescence of the oxidized Alamar blue reagent was measured at times indicated using a Varioskan Flash instrument (ThermoFisher, Waltham, Mass) and normalized to vehicle-treated cells.