Premix wst 1

The control and H₂O₂ treated CEFs, EGK.X blastoderm cells, and PGCs were harvested by centrifugation and washing with PBS. Then, the cells were resuspended in the respective cell culture media containing WST-1 premix (Takara, Tokyo, Japan) at a 1:10 dilution. The complex was aliquated equally to the wells of 96-well plates and incubated at 37 °C, 5% CO₂ for 2 h. The absorbance of the samples against a blank was measured using a VersaMax microplate reader (Molecular Devices, San Jose, CA, USA). Significant differences between the respective control and treated samples were determined by Student’s t test using the GraphPad Prism software (San Diego, CA, USA). Statistical significance was ranked as *P < 0.05, **P < 0.01, ***P < 0.001, or ****P < 0.0001.

Cell proliferation was assessed using the water-soluble tetrazolium salt WST-1 premix (Takara Bio). Cells were seeded at 1 × 10⁴ cells/well in triplicate in two 96-well plates in standard DPSC culture medium, for 0-h and 24-h time points. Medium-only controls were included. The 24-h time point plate was incubated, whereas WST-1 premix was added to each well of the 0-h time point plate at a 1:10 dilution for a total volume of 100 µL, and incubated for 4 h. Absorbance was measured at a test wavelength of 490 nm and reference wavelength of 650 nm, using the GloMax® Discover microplate reader (Promega, USA). This was repeated for the 24-h time point plate the following day. Absorbance was corrected by removing background absorbance (medium-only controls). Proliferation rate was defined as the fold-change in absorbance at 24 h compared to the baseline control (0 h).

The WST-1 proliferation assay was conducted to analyse the proliferation of cancer cells following gas plasma exposure. Therefore, 75 µl of the WST-1 Premix solution (Takara Bio, Japan) was added to each well 22 h after treatment. Following incubation for 2 h at 37 °C and 5% CO₂, the absorbance was measured at 440 nm using a microplate reader (M200; Tecan). Cell-free medium was used for background subtraction. Data were normalised to untreated controls. Cellular viability was further assessed 24 h after gas plasma treatment using flow cytometry. Briefly, cells were stained with 1 µM iFluor 840 maleimide (AAT Bioquest, USA; Cat# 1402) for 20 min at 37 °C. After washing, cells were acquired using flow cytometry (CytoFLEX LX; Beckman-Coulter, Germany) and evaluated using Kaluza 2.1.3 analysis software (Beckman-Coulter).

U2OS, A549, and SKOV-3 cells (5000/well) were seeded in a 96-well plate. Next day, treatment with different concentrations of GPSE extract were given for 48 h. DMSO was used as a solvent control of which the volume was matched to the amount used for respective GSPE- and ARC-treated cells in all the experiments. Cytotoxicity assay was performed using 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay (Life Technologies/Thermo Fisher Scientific, Waltham, MA, USA) as described earlier [23 (link)]. Briefly, viability of control and treated cells was evaluated based on their metabolic activity as determined by the conversion of MTT (yellow) by the mitochondrial NADH dehydrogenases of living cells into formazan (purple). The statistical significance of the results was determined from three independent experiments including triplicate sets in each experiment. For WST assay, cells were treated with different concentrations of GPSE extract for 48 h followed by addition of premix WST-1 (Takara Bio Inc., Shiga, Japan). Cell viability (based on their metabolic activity) was measured at 450 nm with a reference wavelength at 630 nm.

The TNBC cell line MDA-MB-231 was kindly provided by Prof. Moon Hyeong-Gon (Seoul National University Hospital) and cultured in Dulbecco’s modified Eagle’s medium (Hyclone) supplemented with 10% FBS (Hyclone) and 1% antibiotic-antimycotic solution (Gibco). The cells were seeded in triplicate onto 96-well culture plates. After 24 hours (h) of cultivation, the cells were treated with various concentrations of nicotinamide (Sigma-Aldrich) in a dose-dependent manner and Z-VAD-FMK, a pan-caspase inhibitor (Novus). After incubation for 0, 24 and 48 h, the cell viability was assessed using Premix WST-1 (Takara) measured at a wavelength of 450–650 nm.

Cells were seeded into 96-well tissue culture plates at 2×10³ cells per well. At 8, 24, 48 and 72 hours cell viability was determined using the WST-1 assay kit (Premix WST-1®, Takara Bio) following the manufacturer's instructions. The absorbance values at 450nm were normalized by values at 8 hours following subtraction of values obtained from vacant wells.

In order to evaluate the adhesion potential of MDA-MB-231 breast cancer cells, the following adhesion protocol was conducted, as it was described by previous works [39 (link),40 (link)]. Briefly, 96-well plate was coated with collagen type I solution (40 μg/mL) and kept at 4 °C. After 12 h, the solution was removed, and the plate was air-dried; 3% BSA in PBS solution was added in each well, for 30 min, to block non-specific adsorption. Then the solution was removed, and the plate was washed with PBS and air-dried. Cells treated for 24 h prior to the adhesion assay were detached with PBS-EDTA 1× and resuspended in serum-free medium with 0.1% BSA. and seeded at a density of 2 × 10⁴ cells/well. Cells were incubated for 30 min, to allow adhesion to the surface. Non-adherent cells were removed with serum free medium, and then cells were incubated with medium supplemented with 10% FBS for 2–3 h for recovery. After the incubation period, Premix WST-1 (water-soluble tetra-zolium salt) Cell Proliferation Assay System (Takara Bio Inc., Göteborg, Sweden) was added at a ratio 1:10, and the absorbance at 450 nm was measured (reference wavelength at 650 nm).