3.5. Crystal Violet Cell Proliferation Assay

Crystal violet assay was used for both primary and secondary screening of extracts, as described previously [37]. This assay was used to test for the antiproliferative activity of the adherent cell lines. Briefly, each cancer cell line was seeded out in 96-well microtiter plates at a density of 1000 cells/well and incubated for 24 h to allow for attachment to the plate surface. The next day, the stock solution of each extract (20 µg/mL in dimethylsulfoxide) was serially diluted twofold to the desired concentration range, giving a series of five dilutions. Stock solutions and the dilutions were directly diluted 500-fold into the medium. From the working dilutions, 100 μL aliquots were added to each well. DMSO (0.1% (v/v) was used as a solvent. The plates were incubated for an additional 72 h at 37 °C. After 96 h, the culture medium was discarded and replaced with a 1% glutaraldehyde buffer saline for 20 min and then stored under Dulbecco’s buffer solution (pH 7.4) at 4 °C. On the day of staining, the buffer solution was removed, and the cells were stained with 0.02% crystal violet in deionized water (100 µL/well) for 30 min. Excess dye was discarded by washing the plates for 15 min in fresh water. The cell-bound dye was redissolved in 70% (v/v) ethanol/water and the optical density was measured at λ = 570 nm with a Spectramax 384 Plus plate reader (Molecular Devices, Sunnyvale, CA, USA) or a Sunrise plate reader (Tecan; Männedorf, Switzerland). The IC₅₀ values were calculated by linear least-squares regression of the T/C corr values versus the logarithm of the added extract concentration and extrapolating to the T/C corr. values of 50% [38]. The corrected percent growth values (T/C) corr. (%) was calculated with the following equation:

where OD_T is the mean optical density (OD) of the treated cells, OD_C the mean OD of the controls, and OD_C,₀ the mean OD of seeded cells at the time the drug was added.