Mtt working solution

Cell viability was determined by [(3-(4, 5-dimethyl thiazolyl-2)-2, 5-diphenyltetrazolium bromide)] (MTT) assay. Briefly, adherent melanoma cells (SK-MEL-28) and the non-tumoral keratinocyte cell line (HaCat) cells were seeded in 96-well flat-bottomed tissue culture plates at 0.2 × 10⁶ cells/plate in 100 µL DMEM containing 10% FBS and 1% penicillin/streptomycin and various concentrations (5 mM, 8 mM, 12 mM, 15 mM, 18 mM, 20 mM, 24 mM, 28 mM, 32 mM, 36 mM, and 40 mM) of DPG based on a previous study (Bonafé et al., 2019). The cells were cultured for 24 h, 48 h, and 72 h prior to treatment with 100 µL of 0.2 μg/μL of MTT working solution (Sigma, St. Louis, MO, USA) for 4 h at 37 °C. Following incubation, formazan crystals were solubilized with 100 µL of dimethyl sulfoxide (DMSO). Cell viability was determined by measuring the optical density at 550 nm using a microplate spectrometer (Thermo Fisher, Waltham, MA, USA). Cell survival rates were expressed as percentages of the value of normal cells. Untreated control cells were analyzed in all experiments, and all DPG dose treatments were performed in triplicate.

The effect of the different mats on the viability of HeLa cancer cells, SH-4 cells, and mouse BALB/c 3T3 fibroblasts was assessed by the MTT assay [22 (link)]. Briefly, the cells were trypsinized by 0.25% Trypsin-EDTA and were counted using a hemocytometer. Cells were transferred to a 96-well microtiter plate to ensure a concentration of 2 × 10⁴ cells/well. After overnight incubation at 37 °C in a humid atmosphere containing 5% CO₂ to allow cells attachment, the culture medium was replaced and the cells were placed in contact with various mats (CA, CA/5-Cl8Q, CA,PEG, and CA,PEG/5-Cl8Q), preliminarily UV-sterilized for 30 min, and incubated for 24 and 48 h. HeLa cells, SH-4 cells, and BALB/c 3T3 fibroblasts incubated alone and in the presence of 5-Cl8Q were used as controls. Each variant was tested by five measurements. After culturing in the presence of mats, the HeLa, SH-4 cells, and BALB/c 3T3 cells were washed twice with PBS (pH 7.4) and further incubated with 100 μL of MTT working solution (Sigma Chemical) at 37 °C for 3 h; the supernatants were aspirated, and 100 μL of lysing solution (DMSO/ethanol 1:1) was added to each well to dissolve the resulting formazan. MTT assay reading was performed using an ELISA plate reader (TECAN, SunriseTM, Grodig/Salzburg, Austria). Cell viability was calculated as follows:

HeLa cells and SH-4 (Homo sapiens skin melanoma) (from ATCC, Rockville, MD, USA) were cultured in DMEM supplemented with 10% FBS, 100 U/mL penicillin, and 0.1 mg/mL streptomycin in a CO₂ incubator at 37 °C and 5% CO₂. Cell were trypsinized after reaching 80–90% confluence, by 0.25% Trypsin-EDTA and counted with a hemocytometer. Cells were placed in a 96-well plate with a concentration of 1 × 10⁵ cells per well. The medium was changed after overnight incubation at 37 °C in humidified air with 5% CO₂ to facilitate cells attachment. HeLa and SH-4 cells were placed in contact with fibrous materials (CA/PEG and CA/PEG/QUE) for 24 and 48 h. HeLa cells and SH-4 incubated alone and in the presence of QUE were used as controls. MTT assay was used to determine the effect of different fibrous materials on cell viability [25 (link)]. Each variant was assayed by five measurements. After culturing in the presence of mats, the HeLa and SH-4 cells were washed twice with PBS (pH 7.4) and further incubated with 100 μL of MTT working solution (Sigma Chemical) at 37 °C for 3 h. The supernatants were aspirated, and 100 μL of lysing solution (DMSO/ethanol 1:1) was added to each well to dissolve the resulting formazan. MTT assay reading was performed using ELISA plate reader (TECAN, SunriseTM, Grödig/Salzburg, Austria). The percentage of cell viability was calculated as follows:

To analyze the viability of FLSs, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays were used. The FLSs were placed in 96-well plates at an amount of 6,000 cells per well. The cells were cultured for 24 h after transfection. Thereafter, 20 μl/well MTT working solution (Sigma, USA) was added. After 4 h, the liquid was aspirated and 200 μl dimethylsulfoxide (DMSO) was added to each well. Lastly, the absorbance at 490 nm was measured by a microplate reader (Synergy HTX, Biotek, Winowski, VT, USA).