Cell Proliferation Quantification via Crystal Violet

The treatment-induced inhibition of cell proliferation was assessed using crystal violet staining, as described earlier [22 (link)]. In brief, 1500 cells/well seeded in 96-well plates were allowed to adhere to the bottom of the wells for 72 h. Thereafter, the cells were incubated with rising concentrations (0.1–20 μM) of each test compound for up to 48 h. After that, cells were rinsed with PBS, fixed with 1% glutaraldehyde, and 0.1% crystal violet (N-hexamethylpararosaniline, Sigma Aldrich) was added to stain the cells. Unbound dye was removed by rinsing with water. The cell-bound crystal violet was dissolved using 0.2% Triton X-100 (Sigma-Aldrich, Munich, Germany). The extinction of crystal violet, which increases linearly with the increase of the cell number, was measured with an ELISA-Reader (Dynex Technologies, Denkendorf, Germany) at 570 nm [23 (link)].