Traction Force Mapping of Stellate Cells

Polyacrylamide gels containing 0.5 μm diameter fluorescent beads (Life Technologies, Carlsbad, CA, USA) were prepared with a Young’s moduli of 5 kPa using a ratio 7.5:0.175 of acrylamide (40% w/v; Bio-Rad) to bis-acrylamide (2% w/v; Bio-Rad) and then coated with 0.1 mg/mL of rat tail type I collagen (Corning) [55 (link),56 (link)]. HSteCs (20,000 cells) were grown in 35-mm dishes for 24 h, and then large EVs from TJU-UM001 cells (100 µg) were deposited onto adherent stellate cells. After 24 h, phase contrast images of single HSteCs and fluorescent images of the bead field at the surface of the polyacrylamide gel were acquired (Nikon Eclipse Ti microscope, NIS-Elements software) [56 (link),57 (link)]. Cells were removed from the polyacrylamide gel using 0.25% trypsin/EDTA, and a fluorescent image of the bead field was acquired after cell removal [56 (link),57 (link)]. Bead displacements between stressed (+EVs) and null (w/o EVs) states were analyzed for 25 cells per condition using the LIBTRC library to obtain traction stress maps (Pa) and traction force values (nN) [56 (link),58 (link)].