4.8. Labelling of Peptide with Cy5 and Epifluorescence Assay

A solution containing 1:1 molar ratio of AF38Pep to non-sulphated Cy5-NHS-ester (APExBIO, Houston, TX, USA) was incubated overnight in RT, shielded from light and under constant mixing. The following day, the solution was passed through a PD-10 Sephadex G-25 M gel filtration column (GE Healthcare, Chicago, IL, USA) into PBS. The first 3 fractions (highest molecular weight) were collected, dialyzed (membrane MWCO 1 kDa) against ddH₂O overnight, and concentrated by vacuum centrifuge at 3000 rpm at 60 °C for 3 h. Fibroblasts were grown to 70% confluence in 12-well tissue culture plates, before growth arrest in serum-free DMEM for 48 h. Cells were then treated with 10 ng/mL TGF-β1 for 0, 24, 48 and 72 h. The conjugated AF38Pep-Cy5 was added to the plate wells at 10 µg/mL and was incubated for 2 h. The culture medium was removed, and cells were washed three times with PBS before replenishment with fresh serum-free DMEM. Any AF38Pep-Cy5 that remained bound to EDA-FN was assessed using an IVIS imaging system (Caliper Life Sciences, PerkinElmer, Hopkinton, MA, USA) set to detect Cy5.5 epifluorescence after excitation at 640 nm. Epifluorescence was quantified using Living Image v4.2 analysis software (Caliper Life Sciences).