Mtt cell proliferation assay kit

B16-F10 cells (1 × 10⁵) were seeded in 96-well plates with 195 μL of DMEM high-glucose supplemented media (Sigma-Aldrich, 51435C). Primary screening was done to determine IC₅₀ values using the MTT assay kit (Bio-Vision MTT Cell Proliferation Assay Kit #K299-1000) and ProBit analysis after 24 h of exposure. After that, the IC₅₀ of AgNPs (4.2 μg/mL) and Cisplatin (2 μg/mL, CisPt) were employed to determine the cell growth behaviour at 6, 12, 18, and 24 h. The proliferation kinetics was determined by flow cytometry in Attune NxT equipment. Cell viability was determined using the Vybrant™ CFDA SE Cell Tracer Kit and propidium iodide (PI) (Thermo Fisher Scientific, V12883) following the provider's protocol.
Determination of apoptosis and necrosis cell death pathway was determined with the Alexa Fluor® 488 Annexin V/Dead Cell Apoptosis Kit (Thermo Fisher Scientific, V13241). ROS generation was analysed by the DCFDA Cellular ROS Detection Assay Kit (Abcam, 139476) and with the MitoSOX™ Red Mitochondrial Superoxide Indicator for live-cell imaging (Invitrogen, M36008).

Cell proliferation was detected using the MTT Cell Proliferation Assay Kit (Biovision, K299). HK‐2 cells were cultured in a 96‐well plate at a density of 5 × 10³ cells/well. After experimental treatment, 50 µL of serum‐free medium and 50 µL of MTT reagent were added to each well for 3 h at 37°C. Thereafter, 150 µL of MTT solvent was added to each well and shaken on an orbital shaker for 10 min. A microplate reader (BioTek, ELx800, Winooski, VT, USA) was used to measure the absorbance at 570 nm.

MTT Cell Proliferation Assay Kit was purchased from BioVision (Milpitas, CA, USA) and used according to the instructions. HBMEC were seeded in 96 well plates at 5×10³ per well in 100 μL culture medium and incubated for 24 h. Gefitinib was added as indicated in HBMEC binding and invasion assays. Supernatant of each well was removed and MTT dissolved in serum-free medium was added and further incubated for another 4 h. After incubation, 100 μL of MTT solvent was added into each well, and the plate was wrapped in a foil and shaked on an orbital shaker for 15 min. Absorbance of all wells at 570 nm were determined.