Cytotoxicity Evaluation of Murine and Human Cell Lines

The murine melanoma cells (B16-F10), human foreskin fibroblasts (Hs68), and human umbilical vein cell line (EA.hy926) were separately cultured on 96-well plates [28 (link)]. B16-F10 cells (No. CRL-6475) were obtained from the American Type Culture Collection (ATCC: Manassas, VA, USA); Hs68 cells (No. CRL-1635) were purchased from ATCC. Hs68 is one of a series of human foreskin fibroblast lines developed at the Naval Biosciences Laboratory (NBL, Oakland, CA, USA); and EA.hy926 cells (No. CRL-2922) were also acquired from ATCC. The human umbilical vein cell line, EA.hy926, was established by fusing primary human umbilical vein cells with a thioguanine-resistant clone of A549 cell (human lung cancer) by exposure to polyethylene glycol. The testing compound at suitable doses was added to each of the three cell cultures. The cells were cultured in DMEM medium within a 5% CO₂ atmosphere humidified incubator at 37 °C for 24 h. After 24 h incubation, 10% alamarBlue^® (Biosource, CA, USA) was aseptically added to measure cell viability, according to commercial kit protocols. The vehicle control group (without 4-(phenylsulfanyl)butan-2-one) was defined as 100%. The optical absorbance values (A) of the supernatants were quantified at 570 nm, and the cell viabilities were analyzed according to the following formula:
$\text{Cell viability }\left(\text{\%}\right)= \frac{\left(A_{\text{sample}} -A_{\text{blank}}\right)}{\left(A_{\text{control}} -A_{\text{blank}}\right)}\times 100\text{\%}$