Neutral red cytotoxicity test.

The neutral red uptake assay for cell viability was previously described in detail (80) and is adapted here. In brief, 1.5 × 10⁴ BHK-21 cells were seeded into 96-well plates and cultured for 24 h in phenol red-free complete growth medium. Cells were then incubated for 8 h or 24 h at 37°C with serial dilutions of 100% DMSO and 50 mM BBC prepared in phenol red-free medium A (minimal essential medium [MEM], 2 mM glutamine, 0.2% bovine serum albumin [BSA], 100 U/ml penicillin, 100 μg/ml streptomycin, and 10 mM HEPES [pH 7.0], medium S (BHK growth medium with 10 mM HEPES [pH 7.0] and 2% FBS instead of 5%), or 2% FBS medium (medium A with 2% FBS instead of BSA). Cells were then incubated for 2 h with 40 μg/ml neutral red solution in phenol red-free complete growth medium, washed, and extracted, and neutral red absorbance was measured from the top on a SpectraMax M5 microplate reader (Molecular Devices) with SoftMax Pro 7.0 software (Molecular Devices). A dose-response curve with a least-squares (ordinary) fit was drawn using Hill function analysis to determine the concentration of BBC causing 50% inhibition of uptake, which was considered the 50% cytotoxic concentration (CC₅₀) of BBC.