Cell Viability by Crystal Violet Staining

ARPE-19 cells were seeded in a 96-well plate at a density of 2 × 10⁴ cells per well. The cells were incubated at 37°C for 3 days. The medium was removed and replaced with Ringer's solution containing various concentrations of BEL, and incubation continued at 37°C for 3.5 hours. The medium was replaced with complete medium, and the cultures were incubated for a total of 16 hours. After two washes of the cells with deionized H₂O, the plate was inverted and tapped gently to remove excess liquid. A total of 50 µL of a 0.1% crystal violet (Sigma-Aldrich) staining solution in 25% methanol was added to each well and incubated at room temperature for 30 minutes on a bench rocker with a frequency of 20 oscillations per minute. The cells in the wells were briefly washed with deionized H₂O, and the plates were then inverted and placed on a paper towel to air dry without a lid for 10 minutes. For crystal violet extraction, 200 µL of methanol was added to each well, and the plate was covered with a lid and incubated at room temperature for 20 minutes on a bench rocker set at 20 oscillations per minute. The absorbance of the plate was measured at 570 nm.