Figure 2.

Determination of cytotoxicity of MTX. To determine the cytotoxicity of MTX, the number of Vero cells after MTX treatment was directly counted by trypan-blue staining. Then, the number of live cells were counted under a light microscope with a hemocytometer. (A) 10× bright-field images of Vero cells with 0.5 µM MTX treatment with initial cell density of 10,000 cells/100 µL. Scale bars represent 5 µm. (B) The cell density of Vero cells after 0.5 µM MTX treatment after incubation for 48 h at 37 °C and 5% CO₂ (NuAir, Plymouth, MN, USA). (C) Cell viability assay using CTF reagent with initial cell density of 30,000 cells/100µL. Two MTX concentrations (5 µM and 0.5 µM) were tested. At least two independent replicates were performed. One-way ANOVA, followed by Tukey’s multiple comparisons test, were used for statistical analysis. The error bars represent the standard error of the mean (SEM). * p ≤ 0.05.