2.9. HUVEC Culture and MTT Assay

Human umbilical vein endothelial cells (HUVECs; CRL-1730) were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured in Ham’s F-12K medium (Welgene) supplemented with Large Vessel Endothelial Supplement (Thermo Fisher Scientific, Waltham, MA, USA). Cells were seeded into 96-well culture plates (0.5 × 10⁴ cells/well), and while in proliferative stages, were treated with Bu or Cy for 4 h to induce cytotoxic effects followed by CM (secreted from 0.5 × 10⁴ T-MSCs), rhPTN (200 ng/mL), or CM + α-PTN Ab (1 μg/mL) treatment for 16 h. To determine the number of live cells, thiazolyl blue tetrazolium bromide (MTT) was reconstituted in PBS (5 mg/mL), then 20 μL was added to each well. After 4 h of incubation, culture supernatant was removed and 200 μL DMSO was added to elute the formazan production for absorbance measurement at 570 nm.