Ccd 1064sk

MTT assay was applied to evaluate L. enzymogenes CFS cytotoxicity. The cell line used in this assay was human normal skin fibroblast (CCD-1064SK) purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). This colorimetric assay measures the cellular metabolic activity based on the ability of nicotinamide adenine dinucleotide phosphate (NADPH)-dependent cellular oxidoreductase enzymes to reduce the yellow 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide dye (MTT) to form the insoluble formazan purple crystals [48 (link)–50 (link)]. Cells were seeded in a 96-well culture plates in a final volume of 100 μL media per well, then plates were incubated in a humidified atmosphere (37°C, 5% CO₂) for 24 h to allow cells to adhere. When cells reached confluency, they were treated with the L. enzymogenes CFS to obtain final concentrations of 1.25, 2.5, 5, 10, 20, 25, 40, and 50% (v/v). Cells were incubated in the humidified atmosphere (37°C, 5% CO₂) for 72 h before performing the MTT assay to determine the cells’ viability. The assay was conducted in triplicate, and the control cells were treated only with 10% TSB. The optical density for the treated and control wells was measured at 570 nm using a microtiter plate reader (BioTek, Winooski, VT, USA). Percentage viability was calculated using the formula below
$$\mathrm{\%}\mathrm{V}\mathrm{i}\mathrm{a}\mathrm{b}\mathrm{i}\mathrm{l}\mathrm{i}\mathrm{t}\mathrm{y}=\left(\frac{\mathrm{M}\mathrm{e}\mathrm{a}\mathrm{n}\mathrm{O}\mathrm{D}\mathrm{s}\mathrm{a}\mathrm{m}\mathrm{p}\mathrm{l}\mathrm{e}}{\mathrm{M}\mathrm{e}\mathrm{a}\mathrm{n}\mathrm{O}\mathrm{D}\mathrm{b}\mathrm{l}\mathrm{a}\mathrm{n}\mathrm{k}}\right)\times 100$$

Cell lines of melanoma A375 (CRL-1619), G-361 (CRL-1424), and SK-MEL-1 (HTB-67) from the Melanoma Cancer Cell Panel (TCP-1013) and the cell line of normal fibroblasts CCD-1064Sk were obtained from the ATCC (American Type Culture Collection, Manassas, VA, USA). The A375, G361, SK-MEL-1, and CCD-1064Sk cells were cultured accordingly in Dulbecco’s Modified Eagle Medium (DMEM; Gibco, Thermo Fisher Scientific, Waltham, MA, USA), McCoy’s 5A Modified Medium (Sigma-Aldrich, St. Louis, MO, USA), Eagle’s Minimum Essential Medium (EMEM, Gibco, Thermo Fisher Scientific, Waltham, MA, USA), and Iscove’s Modified Dulbecco’s Medium (IMDM, Gibco, Thermo Fisher Scientific, Waltham, MA, USA). All the media were supplemented with 10% fetal bovine serum (FBS; Gibco, Thermo Fisher Scientific, Waltham, MA, USA) and 1% antibiotic/antimycotic solution (Gibco, Thermo Fisher Scientific, Waltham, MA, USA). The cells were incubated under standard culture conditions (37 °C, 5% CO₂, and relative humidity 95%).

The cell line under investigation was normal human skin fibroblast cells (CCD-1064SK) purchased from American Type Culture Collection (ATCC) (Manassas, VA, USA). Fibroblast cells were cultured in Iscove’s Modified Dulbecco’s Medium (Euro Clone, Italy) as recommended by ATCC. The media was supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Ebsdorfergrund, Germany), 1% of 2 mM L-glutamine, 100 U/mL penicillin, and 100 µg/mL streptomycin. According to the cells’ growth profile, fibroblast-seeding density was 1 × 10⁵ cell/well. Cell viability was determined by trypan blue exclusion using a hemocytometer.