Celltiter glo 3d viability assay

To determine cell viability in 2D culture, equal numbers of cells were seeded into 12-well plates. Subsequently, cells were treated with DMSO, tunicamycin (4 μg/ml), or irinotecan (10 μM) for 48 h in complete growth media. The cell viability was determined either by counting the number of live cells using an automated cell counter (Beckman-Coulter) or by staining with the alamarBlue Cell Viability Reagent (Thermo). The relative cell survival was calculated by normalizing numbers of surviving cells in the drug-treated group to that of the DMSO-treated group.
To determine cell viability in 3D culture, equal numbers of cells were mixed with 50% Matrigel in complete growth media and seeded into 24-well plates. Subsequently, cells were treated as described above for 48 h. The relative cell survival was determined using the CellTiter-Glo 3D viability assay (Promega) and normalized to the DMSO treatment group.

Spheroids ATP content was determined with Cell-Titer Glo 3D viability assay (#G9681, Promega, France), according to the manufacturer’s protocol. Briefly, 5 minutes after illumination, the spheroids were rinsed in a large volume of PBS. Eighty microliters of PBS containing one spheroid were then deposited per well in a white 96-well plate, and 80 μL of the reagent were immediately added to the wells. Spheroids were incubated under gentle rotation for 30 min at room temperature to allow tissue lysis. ATP content was measured by light emission using a luminometer (Clariostar, BMG Labtech, Germany).

Organoids were propagated in hPDAC media modified from Boj et al.^{66 (link)}. For drug screening, organoids were dissociated to single cells and seeded on top of a thin layer of Matrigel (Corning, NY, USA) in 384-well plate (3000/well). Next day, organoids were treated with a range of MK-2206 (0.05–30 µM) and Trametinib (0.001 nM to 10 µM) or SHP099 (0.05–400 µM) concentrations for 96 h and cell viability was determined by CellTiter-Glo 3D viability assay (Promega, Madison, USA). Drug-response curves were graphed and half maximal inhibitory concentration values were calculated using GraphPad Prism 8.0 (San Diego, USA).

Anti-cancer compounds and low toxicity compounds listed in Additional file 3: Table S2 were purchased from Selleck Chemicals. Dissociated organoid cells were plated in Medium/Matrigel at 3500 cells/well in triplicate 72 h before drug treatment. Organoids were treated for 6 days in a humidified atmosphere at 37 °C, 5% CO₂ and drug-containing medium was replaced every 72 h. Cell viability was determined with CellTiterGlo 3D viability assay (Promega). Further details are provided in Supplementary Methods, Additional file 1.

The CellTiter-Glo 3D Viability-Assay (Promega, Southampton, UK) was mixed with media at a ratio of 1:1 and then incubated 5 min on a plate shaker and 25 min on the benchtop with light protection. Measurements were taken in triplicate using the Tecan Infinite Lumi plate reader (Männedorf, Switzerland). All values were normalised to media control readings.

Viability was assessed using the CellTiter-Glo 3D Viability assay (Promega, cat. No. G9681, Walldorf, Germany). Spheroids and surrounding medium were collected after four days and transferred to an opaque-walled 96-well plate. A volume of CellTiter-Glo Reagent equal to the volume of cell culture medium was added. The mix was incubated according to manufacturer instructions and luminescence in the form of relative light units (RLUs) was recorded using a CLARIOstar Plus (BMG Labtech, Ortenberg, Germany).

1,2-Dimyristoyl-sn-glycero-3-phosphocholine (DMPC), N-(carbonyl-methoxypolyethylenglycol-2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine, and Na-salt (DSPE-PEG 2000) were purchased from Lipoid, Ludwigshafen, Germany. Baicalein was purchased from Haoxuan Biotech, Xi’an, China. Chloroform, methanol, and tert-butanol were purchased from Stanlab, Lublin, Poland. Sodium chloride, dimethylsulfoxide (DMSO), and ethylenediaminetetraacetic acid (EDTA) were purchased from ChemPur, Piekary Śląskie, Poland. Sepharose 4B CL, thiazolyl blue tetrazolium bromide, 2-(4-amidinophenyl)-6-indolecarbamidine dihydrochloride, and 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) were purchased from Merck, Darmstadt Germany. The DiL stain (1,1ʹ-dioctadecyl-3,3,3’,3’-tetramethylindocarbocyanine perchlorate) was purchased from Thermo Fisher, Waltham, USA. The RPMI-1640 and MEM-Alpha cell culture media were purchased from Lonza, Basil, Switzerland. Fetal bovine serum (FBS), GlutaMAX (L-alanyl-L-glutamine dipeptide in 0.85% NaCl), and 100× antibiotic-antimycotic were purchased from BioWest, Nuaillé, France. Carbon films were purchased from Lacey Formvar/Cu grids, SPI Supplies, West Chester, PA, USA. Agarose was purchased from Roth, Karlsruhe, Germany. The CellTiter-Glo 3D Viability Assay and RealTime-Glo Annexin V Apoptosis and Necrosis Assay were purchased from Promega, Madison, WI, USA.