4.7. Neutral Red Uptake Assay

Cells were seeded in 100 μL of media at a density of 10⁴ cells/well in 96-well microtiter plates. Solutions of compounds were previously prepared in DMSO, and 1 μL of the corresponding solution was added to each well. The final volume of each well was adjusted to 200 μL. After 72 h of incubation, culture media were removed, and 100 μL of 10 μg/mL neutral red solution prepared in culture media was added to each well and incubated for 2 h at 37 °C in a humidified atmosphere containing 5% CO₂. Then, media were aspirated, the plate was washed three times with PBS 1X, and 100 μL of neutral red distain solution (50:49:1, ethanol/water/glacial acetic acid) was added. The plate was placed for 15 min in a shaker, and fluorescence was measured using Cytation 5 apparatus (Biotek, Winooski, VT, USA) at 530/645 nm excitation/emission wavelengths.