Luminescent cell viability assay kit

The CP70 and SKOV3 ovarian cancer cell lines were seeded at a density of 2 × 10³ cells per well in a 96-well plate in fresh culture medium. Next, following a scheduled time course, we added the nano-NI to each well to obtain a final concentration of 3 μM in the test wells. Then, the luminescent cell viability assay kit (Promega) was used to collect the supernatant for laminator analysis, the measurements were performed after 15 min using a microplate reader, and then calculated using the Gen5 software (BioTek).

MG63 cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and maintained in a humidified incubator (37 °C, 5% CO₂) with α-MEM (Sigma-Aldrich) containing 10% fetal bovine serum. Cells (1 × 10⁶) were seeded into a 100-mm diameter cell culture plate and maintained for 3–4 days, after which 1 × 10⁵ cells were seeded onto each scaffold in a 96-multi-well white plate and later counted using a Luminescent Cell Viability Assay Kit (Promega, Madison, WI, USA). To evaluate cell differentiation, 1 × 10⁵ cells were seeded in a 24-well cell culture plate and calcium deposition was measured. Bone differentiation was evaluated by Titrate-Resistant Acid Phosphatase and ALP Double-Staining Kit (Takara, Shiga, Japan). ALP activities were determined by absorbance measurements at 405 nm, with a microplate reader (Bio-Tek instruments, Winooski, VT, USA) and normalized to the protein concentration [29 (link)].

The effect of the ADBR2-blockade on the cell viability of primary ccRCC tumors and 786-O cells was performed by analyzing the number of viable cells in the culture, based on the quantification of the presence of ATP, hence indicating metabolically active cells.
First, 5 × 10³ cells/well were seeded in a 96-well plate and incubated in complete medium with [0- 50- and 100-μM] propranolol or ICI for 72 h. Then, 100 μL/well of the Luminescent Cell Viability Assay kit (Promega, Madison, WI, USA) was added and gently mixed for 15 min at RT. Luminescence was measured using a GLOMAX multidetection apparatus (Promega).