Brdu colorimetric cell proliferation elisa

The proliferation assay was performed with previously serum-starved cells using a BrdU colorimetric cell proliferation ELISA (Roche, 11647229001), according to the manufacturer’s protocol, and measured using a microplate reader (Infinite M200 PRO; Tecan, Männedorf, Switzerland). For the migration assay, a similar number of cells was seeded into a Transwell membrane insert with reduced medium (8-µm pore size; Falcon, 353097, BD) and allowed to migrate for 6 h toward the lower compartment containing the complete medium. Thereafter, migrated cells were fixed with methanol, stained with 10% crystal violet (Sigma-Aldrich, V5265), excised, and then fixed onto slides using Pertex mounting medium (Medite Service AG). Slides were scanned using a slide scanner (NDP Nanozoomer 2.0HT). and quantified with Fiji software and ITCN macro (NIH, Bethesda, MD, USA).

Proliferation assays were performed with serum-starved cells exposed for 24 h to RPMI-1640 supplemented with 1.5 mg/ml Pirfenidone using a BrdU colorimetric cell proliferation ELISA according to manufactures instructions (Roche, Basel, Switzerland). Absorbance was measured at 370 nm with reference at 492 nm in a plate reader (TECAN, Männedorf, Switzerland). The proliferation of cells was plotted as a percentage of absorbance compared with the control cells' absorbance. At least three independent experiments were performed with 8 technical replicates each.