4.4. Determination of Cell Viability by Neutral Red Uptake Assay

Drug induced cytotoxicity in HFFs was measured as a reduction of cell viability determined by neutral red uptake assay (NRA), as described previously [62,63]. Briefly, HFFs treated with compounds for 7 d were incubated with a final concentration of 40 μg/mL neutral red (Sigma Aldrich, St. Louis, MO, USA) for 3 h. Incorporated neutral red was released from the cells by incubation with destaining solution (50% ethanol, 49% H₂O, 1% acetic acid) and subsequently quantitated in a microplate reader (PerkinElmer, Waltham, MA, USA) by fluorescence measurement using 560/630 nm for excitation/emission, respectively.