Elisa microplate reader

Cells were plated in triplicates in normal medium in 96‐well plates. Cell viability was assessed at the indicated time points by incubating cells with medium containing MTT (3‐(4,5‐dimethylthiazolyl‐2)−2,5‐diphenyltetrazolium bromide) solution (0.5 mg mL⁻¹; Sigma) for 2 h at 37 °C. Cells were then lysed in 100 µL lysis buffer (Isopropanol containing 0.4% NP‐40 in 0.04 mol L⁻¹ HCl), and absorbance was measured using ELISA microplate reader (Corning, NY, USA) at 570 nm with a 620 nm reference wavelength.

For cell viability assays, cells were plated in 96-well plates at the indicated numbers in triplicates. Cell viability was assessed after 24 and 72 h by MTT (3-(4,5-dimethylthiazolyl-2)−2,5-diphenyltetrazolium bromide) colorimetric assay. The cells were incubated with medium containing MTT solution (0.5 mg/ml; Sigma) for 3 h at 37 °C. Cells were lysed with 100 μl lysis buffer (0.4% NP-40 in 0.04 mol/l HCl-isopropanol), and absorbance was measured at 570 nm with a 680 nm reference wavelength using ELISA microplate reader (Corning, NY, US). Cell viability is depicted as fold growth at 72 h compared to 24 h. The number of seeded cells to obtain low and high densities in different TNBC cell lines were as follows: HCC1937—low: 1.5 × 10⁴ ml⁻¹; high: 1 × 10⁵ ml⁻¹, MDA-MB-468—low: 2 × 10⁴ ml⁻¹; high: 1.6 × 10⁵ ml⁻¹, Hs578T—low: 1 × 10⁴ ml⁻¹; high: 7.5 × 10⁴ ml⁻¹, BT549—low: 1 × 10⁴ ml⁻¹; high: 7.5 × 10⁴ ml⁻¹.