Celltiter glo 3d cell viability assay

Cell culture viability was measured by using the CelltiterGlo 3D Cell Viability Assay (#G9681, Promega, Charbonnières-les-Bains, France), which measures the quantity of ATP via luciferase activity. This decrease could be correlated with a decrease in cell viability, cell metabolism, or cell number. Treated cells were left for the desired time point prior to the addition of 110 µL CelltiterGlo 3D reagent. Wells were then periodically flushed vigorously via pipetting in order to disaggregate spheres and encourage lysis prior to transfer to a white opaque 96-well culture plate (#236105, ThermoFisher, Illkirch-Graffenstaden, France) to prevent luminescence leakage between wells during relative luminescence units (RLU) counting using a plate reader (SP2000 Safas, Monaco). Results were expressed by the subtraction of background RLU (culture medium and assay reagent) and expression of viability relative to non-treated controls (considered as 100% viability). The percentage of viability was calculated using the following equation (Equation (1)):
IC50 values were calculated using GraphPad Prism software from dose–response curves using non-linear regression (log(concentration) vs. %viability—variable slope (four parameters)).

The CellTiter Glo^® 2.0 and CellTiter Glo^® 3D cell viability assay systems (Promega, Madison, WI, USA) were used to quantify cytotoxicity by measuring metabolic active cells through ATP presence through a luminescent luciferase reaction that is directly proportional to viable cells in the culture. Briefly, target cells and effector cells were co-cultured in the indicated E:T ratios, cell numbers, treatments, and time points. At the experimental end point, the viability assay reagent was added to the co culture in a 1:1 ratio (total culture volume–assay reagent) followed by shaking of the assay plate, incubation for 10 min (2D) or 25 min (3D) at room temperature, and subsequently measured using a Tristar 3 multimode plate reader (Berthold Technologies, Bad Wildbad, Germany). Target cell lysis was quantified using the formula: $$\mathrm{Cytotoxicity} (\%)=100-(\frac{Sample release-Effector cell release}{Maximum release})\times 100$$

The Iso-50 organoids in the culture were gently dissociated to the near single-cell population using TrypLE (Gibco, Life Technologies, Renfrew, Scotland, UK) before resuspension within a growth-factor reduced Matrigel. The individual cells were counted using a Trypan blue exclusion test (Gibco, Life Technologies, UK) and were seeded at a range of densities (from 100 to 1000 cells/µL of Matrigel dome) into a 24-well plate or microfluidic device (30 µL blob per well). The ATP measurement of the CellTiter-Glo 3D Cell Viability Assay (Promega, Madison, WI, USA) was then performed to evaluate the organoid proliferative ability according to the manufacturer’s instructions.

Total intracellular adenosine triphosphate (ATP) content was quantified by luminescence using CellTiter-Glo® 3D Cell Viability Assay (Promega, Madison, WI, USA) with a slightly modified protocol. In brief, for each replicate one individual spheroid was transferred by sedimentation into a white 96-well assay plate (3903 Corning, Corning, NY, USA) containing 50 µl PBS (w/o Mg²⁺, Ca²⁺) per well. Subsequently, 50 µl of assay reagent were added following vigorous mixing for further promoting penetration of the 3D reagent into the spheroid matrix. The plate was incubated in the dark for 25 min at room temperature and luminescence was read using a SpectraMax Gemini XS (Molecular Devices, San Jose, CA, USA) reader.

After trypsinization, cell dilutions were re-suspended in media containing 10% FBS and collagen (Stemcell Technologies) on ice. Cells were then plated in U-bottomed 96-well plates (Corning) at 3000 cell/well with 0.015 mg/well collagen. After 24 h, single or multiple viable spheroids were generated. On Day 2, 4 and 6, spheroids were imaged by fluorescence microscopy (Olympus Corporation) using a 10× objective. Cell viability was measured with the CellTiter-Glo® 3D Cell Viability Assay (Promega), following the manufacturer's recommendations. Luminescence was measured at room temperature using a Synergy H1 Hybrid Multi-Mode Microplate Reader (BioTek Instruments, Inc).

For in vitro drug treatments, 4T1 cells were seeded in DMEM containing 10% FCS and 30% Matrigel Growth Factor Reduced (Corning, 356,231) at 300 cells per well in 384-well plates in quadruplicates. After three days, 3D colonies were treated with DMEM containing 1% FCS together with CLR457, SHP009, Sunitinib or combinations thereof at the indicated concentrations. Two days later, 50% of the culture medium was exchanged with medium containing drugs at 200% higher concentrations. Cells were kept under treatment for a further two days. At treatment day 5, the viability of cells was assessed by the CellTiter-Glo 3D Cell Viability Assay (Promega, G9618) according to the manufacturer’s instructions. In brief, after removing the culture medium, cells were lysed in 25 µl CellTiter-Glo 3D Reagent. After a 30-min incubation at room temperature on a horizontal shaker, luminescence was recorded for 0.5 s with an ELISA-reader.