Cytotoxicity ldh assay kit wst

GO/BAC was prepared on glass-based dishes (AGC Techno Glass Co. Ltd., Haibara, Japan) for fluorescence staining, and the bottom of a 96-well culture plate (for cell viability and cytotoxicity assays) by the same method used for the turbidity assessments (with slight modification of the GO and BAC liquid volume). Fibroblastic NIH3T3 cells (1 × 10⁴, RIKEN BioResource Center, Tsukuba, Japan) and culture medium (MEM alpha, GlutaMAX-I; Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (Qualified FBS; Thermo Fisher Scientific) and 1% antibiotics (penicillin–streptomycin; Thermo Fisher Scientific) were inoculated on each substrate to examine its biocompatibility properties. After 24 h of incubation at 37 °C in a 5% CO₂ environment, the cultured cells were fluorescently stained using a LIVE/DEAD Viability/Cytotoxicity kit for mammalian cells (Thermo Fisher Scientific) and observed using fluorescence microscopy. Furthermore, cell viability and cytotoxicity were examined using a water-soluble tetrazolium salt (WST)-8 assay kit (Cell Counting Kit-8, Dojindo Laboratories, Mashiki, Japan) and a lactate dehydrogenase (LDH) assay kit (Cytotoxicity LDH Assay Kit-WST, Dojindo Laboratories), respectively. The absorbance at 450 nm (WST-8 activity) or 490 nm (LDH activity) was measured using a microplate reader (Multiskan FC, Thermo Fisher Scientific).

Cultured hPASMCs were treated with DPP4 siRNA or control siRNA, detached using ACCUTASE (Thermo Fisher Scientific), and cultured in serum-free medium for 24 h and subsequently challenged with TGFβ or PBS. For the proliferation assay, the Cell Counting Kit-8 (WST-8. Dojindo Molecular Technologies. Kumamoto, Japan) was used according to the manufacturer’s protocol. In brief, the treated cells were added to a 96-well plate, 10 μL of WST-8 was added to each well, and the plate was incubated at 37 °C for 2 h. Cell viability was determined by measuring the absorbance at 450 nm using a microplate reader. For the cytotoxicity assay, a Cytotoxicity LDH Assay Kit-WST (Dojindo Molecular Technologies) was used to measure LDH expression levels according to the manufacturer’s protocol. The treated cells were added to a 6-well plate and 100 µL of LDH substrate solution was added to the wells, followed by incubation of the plates for 30 min at room temperature. The absorbance at 450 nm was measured using a microplate reader.

Cells were seeded in 96-well plates at a density of 5 × 10³ cells/well and incubated for 24, 48, 72, and 96 h. The concentration of lactate in the cell culture supernatants at each time point was determined using a lactate assay kit-WST (DOJINDO, Kumamoto, Japan) according to the manufacturer’s instructions. Additionally, LDH activity was determined using a cytotoxicity LDH assay kit-WST (DOJINDO), according to the manufacturer’s instructions.