Neutral Red uptake assay.

Vero cells were seeded into 24-well trays at a density of approximately 4 × 10⁴ cells per well and incubated overnight at 37°C in 5% CO₂. Cell monolayers were preincubated with or without various inhibitors for 1 h and then pulsed with 10 μg/ml ArtB for 30 min, washed twice with PBS, and incubated in fresh medium for another 3.5 h. The medium was then removed, and cells were incubated for 1 h in fresh medium with 400 μg/ml Neutral Red (MP Biomedicals). Cells were washed twice with PBS and then agitated in 300 μl 50% ethanol–1% acetic acid for 10 min, after which 200-μl volumes were transferred into 96-well-flat bottom trays and the absorbance at 540 nm was measured by using a SpectraMax M2 microplate reader (Molecular Devices).