HGF Attachment and Proliferation Assay

HGFs attachment and growth was assessed on all surfaces using CyQUANT^® Cell Proliferation Assay Kit (C7026; Molecular Probes, Eugene, OR, USA) according to the manufacturer’s instructions and our previous experience [24 (link)]. Briefly, in parallel cultures, 10,000 (Attachment assays) or 5000 (Proliferation studies) HGFs was seeded onto each disc and at each designated timepoint, media was removed gently with a pipette gun, followed rinse by cold PBS twice to remove unattached cells. A total of 5000 cells were seeded for proliferation to have significant surfaces area for growth as HGFs are contact inhibited. Lysates were then transferred into a new 24-well plate and stored at −80 °C. DNA content was determined by performing CyQUANT^® Cell Proliferation Assay Kit (C7026; Molecular Probes, Eugene, OR, USA). Cell numbers were extrapolated using a standard curve. Three independent experiments were performed, with 6 replicates per condition per experiment. For attachment, timepoints selected were 1, 6 and 24 h, and for cell proliferation, 1, 3 and 7 days.