Celltiter glo 3d luminescent cell viability assay

COMI cells grown as gliomaspheres were mechanically dissociated to single cells, counted, and plated at a density of 1000 cells per well in Ultra-Low attachment round bottom 96-well plates (Costar). Cells were incubated for 3–4 days to allow the formation of a gliomasphere. Then, cells were treated with different concentrations of 7 or ATO and incubated for additional 4 days. Each treatment was performed in a technical quadruplicate. At the end of the treatment, spheres were stained with 1 µM Calcein AM (Thermo Fisher Scientific, Waltham, United States, cat. C3100MP) and 0.02 µM SYTOX™ Red Dead Cell Stain (Thermo Fisher Scientific, Waltham, United States, cat. S34859) for 1 h at 37 °C in the dark and images were acquired with the MolecularDevices ImageXpress Micro Confocal High Content Imaging System. Afterward, viability was determined using the CellTiter-Glo^® 3D Luminescent Cell Viability Assay (Promega, Madison, WI, United States, cat. G7570) as per the manufacturer’s instructions. Viability was calculated as the percentage of the non-treated control.

Different concentrations of the tested compounds (12.5 to 100 μM) in 100 μL medium were added to the SH-SY5Y and SK-N-AS spheroids. The control wells contained solvent ≤1% v/v. The CellTiter-Glo^® 3D Luminescent Cell Viability Assay (Promega^®, G968B, Milan, Italy) was used to measure intracellular ATP levels. After 72 h, the medium was removed, and CellTiter-Glo reagent (50 μL) was added. Cell lysis to extract ATP was performed by vigorous mixing (5 min). This was followed by 25 min of incubation at r.t. protected from light, transferring the supernatant to an opaque, white, flat-bottomed 96-well plate, and measuring luminescence using Tecan.

Colon normal organoids (passages 8–13) were disaggregated in 1:4 TrypLE Express to single cell and 48,000 cells were seeded on 30 µl Matrigel (10 µl/drop) in 24-well culture dishes. Organoids were cultured in PROL medium plus Y27632 for 5 days. Then, organoids were washed twice in PBS and incubated in differentiation medium (DIFF, BMP4, DBZ or B + D) in the presence of 100 nM calcitriol or vehicle (ethanol). Medium and calcitriol/vehicle were replaced every other day. Upon 5 or 7 days of incubation of organoids in differentiation medium, cell viability was determined by estimating the amount of cellular ATP using the Cell Titer-Glo 3D Luminescent Cell Viability Assay (Promega) following the manufacturer’s instructions.

Cell viability was assessed by determining the ATP content of PHH cultured in 3D with the CellTiter-Glo 3D Luminescent Cell Viability Assay (Promega).

For assay of cell viability, B16-F10, H1703, A549 and NIH-3T3 cells were plated in 96-well round-bottomed plates at a density of 2 × 10³/well and incubated for 72 h with various concentrations of nintedanib. Cell viability was then assessed with the use of a CellTiter-Glo 3D Luminescent Cell Viability Assay (Promega). For assay of cell proliferation, B16-F10 or NIH-3T3 cells were seeded in 10-cm dishes and exposed to 0.5 or 1 μM nintedanib for 24, 48 or 72 h. The cells were then washed three times with ice-cold PBS, collected with the use of Accutase solution (BD Biosciences), and stained with the use of a Zombie Fixable Viability Kit (BioLegend) for discrimination of live from dead cells. The living cells were counted with a flow cytometer (LSRFortessa X-20, BD Biosciences) and normalised by those in untreated cultures.