Mtt cell proliferation viability assay kit

P-MCs, PDE-FBs, and P-FBs were seeded into 96-well plates at a density of 4 × 10³ cells/well. MTT assays were performed on 30 v/v% PDE, 10 ng/mL PDGF-B (R&D Systems, USA), or 10 ng/mL TGF-β (R&D Systems, USA)-treated P-MCs, PDE-FBs, or P-FBs (n = 5 well/treatment group) in the absence or presence of 4.5 × 10⁹–10¹⁰ particles/mL PDE-EVs according to the instructions of the manufacturer (MTT Cell Proliferation/Viability Assay kit, R&D Systems, USA). Absorbance was recorded at 570 nm and at 690 nm as background in a SPECTROstar Nano microplate reader using SPECTROstar Nano MARS v3.32 software (BMG Labtech, Ortenberg, Germany). Vehicle-treated cells (4 mM hydrogen chloride (HCl) in case of PDGF, TGF-β, 30 v/v% 1.5% glucose-containing peritoneal dialysis fluid (Fresenius Medical Care, UK) in the case of PDE and PBS in the case of EV) served as controls. Results were normalized and determined as the percentage ratio of control group values.

Cell proliferation was monitored by a 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) Cell Proliferation/Viability Assay kit (R&D PUBLIC "-//NPG//DTD XML Article//EN"S) in according to the guidelines.

To assess the effect of direct irradiation with NTAPP on the proliferation of MC3T3-E1 cells, an MTT assay was conducted for each experimental condition using an MTT Cell Proliferation/Viability Assay kit (R&D Systems, Minneapolis, USA) following the manufacturer’s specifications. Briefly, 1.8 ×10⁵ cells from cell culture passage five were seeded onto a 24-well plate with-MEM containing 10% FBS. After cultivation for 24 h to allow for sufficient cell attachment, the medium was replaced, and NTAPP was irradiated to the fresh medium surface. NTAPP irradiation was conducted once, and irradiation duration was relatively shorter at 5, 10, and 15 s, compared to the animal study, which was 5 min and 15 min. Two control groups were defined: untreated and irradiated with helium gas for 5 s. After incubating for 24 h and 48 h, the MTT assay was performed. The absorbance was measured at a fixed wavelength of 570 nm using a microplate reader (U-3000 spectrophotometer; Hitachi, Tokyo, Japan). Each group was assessed in triplicates.

In vitro, measurement of cell viability was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) cell Proliferation/Viability Assay Kit (R&D Systems) according to the manufacturer’s specifications. HS-SY-II was seeded in a 10 cm plate and had grown to approximately 100% confluence. Next, HS-SY-II was digested with 0.25% trypsin and 0.02% EDTA and centrifuged at 1200 rpm for 3 min and was plated at a density of 6.4 × 10⁴ cells per well in 96-well plates. The cells were cultured for 24 h before treatment and then exposed to ethanol for 5 min, washed once with phosphate-buffered saline (PBS). The MTT assay was conducted with five concentrations of ethanol (i.e., 0%, 10%, 20%, 30%, and 99.5%). After exposure to ethanol, an MTT assay was performed. Absorbance at 570 nm was determined using a microplate reader (Varioskan LUX; Thermo Fisher Scientific, Inc.).