3.5.6. Cell Death Pattern through Acridine Orange/Ethidium Bromide (AO/EB) Test

In 24-well plates, CALU-3 cells were seeded onto coverslips at the concentration of <sup>1</sup> <sup>×</sup> 105 cells.well−<sup>1</sup> using RPMI 1640 culture medium. After 24 h for cell adhesion, the medium was then discarded and NCUR/MTX-2 sample (CUR 9.88 <sup>μ</sup>mol·L−<sup>1</sup> and MTX 4.00 <sup>μ</sup>mol·L−1) was added in a volume of 500 μL. Cells were then incubated at 37 ◦C in a wet atmosphere and 5% CO2.

After 24 h treatment, the wells were washed with 200 μL of sodium phosphate buffer at pH = 7.4. Following this, 10 <sup>μ</sup>L of acridine orange/ethidium bromide dye mixture (200 <sup>μ</sup>g·mL<sup>−</sup>1) was placed on a coverslip, which was then inverted on a slide. The staining was viewed under a BX41 fluorescence microscope (Olympus, Tokyo, Japan) with an excitation filter at 480/30 nm and emission at 535/40 nm. The typical fields were recorded with an attached Olympus DP71 camera.

Classification of cell type to AO/EB staining was performed based on the criteria proposed by Ribble et al. [60]. In summary, the viable cells present bright green nucleus, necrotic cells depict orange-red fluorescence, early-stage apoptotic cells are marked by crescent-shaped or granular yellow-green AO nuclear staining, and late-stage apoptotic cells show concentrated and asymmetrically sited orange nuclear EB staining.

#### *3.6. Statistical Analysis*

Statistical analyses were performed using one-way analysis of variance (ANOVA), and when necessary, the post-hoc Tukey test was used. The results were expressed as a mean ± standard error of the mean (SEM) and mean ± standard deviation (SD). P values lower than 0.05 (*p* <0.05) were considered significant. Calculations were carried out using the statistical software GraphPad Prism version 5.03 (GraphPad Inc., San Diego, CA, USA).
