Fibroblast Transfection and Hydrogel Photoresponsive Assay

NIH/3T3 fibroblasts were transfected with an αSMA-luciferase and Postn-luciferase promoter plasmids^{[72 (link),74 (link)]}. Transfection conditions (1.66 μg DNA/μL Lipofectamine 3000) were selected so as to maximize transfection efficiency (here, 65%) while retaining cell morphology and health. Transfected NIH/3T3 fibroblasts were encapsulated (5 × 10⁶ cells mL⁻¹) in 50 mol% LOV2-Jα hydrogels in three polystyrene 24-well cell culture plates (Corning) under three different material conditions: 1) continuous flood exposure (λ = 470 nm, 1 mW cm⁻²) giving rise to softer gels; 2) dark culture yielding comparatively stiff materials; and 3) shuttered light exposure (1 min on, 4 min off). After cultured maintenance of these light exposure conditions for 48 h, samples were ground with a mortar and pestle and resuspended in lysis buffer (50 μL, 1% Triton, 100 mM Tris-HCl, 2 nM EDTA, 2 mM DTT) prior to luciferase analysis (Pierce Firefly Luciferase Glow Assay, Thermo Fisher). Luciferase activity was measured in triplicate for each material condition.