Celltitre glo 3d

Patient-derived organoids were obtained from resected tumours of patients with colorectal cancer. All patients provided informed consent before tumour collection, and the study was approved by the Human Research Ethics Committee (HREC 2016.249). Organoids were grown in DMEM/F-12 media with Matrigel (Corning) and seeded in 384 well plates [34 (link)]. After 72 hours of establishment, the organoids were treated with equimolar concentrations of BZA, 5-FU, OX and SN-38 (50 μM - 0 μM) for 7 days. Cell viability was determined using CellTitre-Glo 3D (Promega) according to the manufacturer’s instructions using the EnVision Plate Reader (PerkinElmer). Fluorescence readouts were converted into relative activity using DMSO as a negative control (vehicle treatment) and 1 μM bortezomib as a positive killing control.

2D and 3D cells were seeded as per cell viability assay. 24 hours after seeding, 2D and 3D cells were treated with the same fixed concentrations of neratinib and docetaxel. Approximate IC₅₀ concentrations of drugs were used as established previously in our lab by the acid phosphatase method. Concentrations of neratinib used were: 2.5nM – BT474 cells, 49nM – HCC1954 cells and 6.8nM – EFM192A cells. Concentrations of docetaxel used were: 2.8nM – BT474 cells, 0.6nM – HCC1954 cells and 2.6nM – EFM192A cells. 5 days after treatment, cell viability was determined by measuring cellular ATP levels using CellTitre-Glo 3D (Promega).

Freshly isolated acinar cells were incubated for 30 min with supplemented Medium-199 in 48-well plates (Greiner Bio-One, Hungary), then AZA containing (0.1–1-10–100-1000 μg/mL) media was added, and cells were incubated for another 60 min at 37 °C. Cerulein (100–1000 nM) was applied as positive control. We determined the intracellular ATP content, which is proportional to the number of viable cells, using the CellTitre-Glo 3D (Promega) luminometric assay on a CLARIOStar plate reader. The total protein amount was determined using by Bradford assay in Spectrophotometer (Thermo Scientific™ NanoDrop™ One). Blank-corrected data were normalized to the total protein amount.
In addition, living, apoptotic, and necrotic cells were stained in 8-well chamber slides (Sarstedt, Germany) with Apoptosis-Necrosis Kit (Abcam) and were visualized under a Zeiss LSM880 confocal microscope using a 40× oil immersion objective (Zeiss, NA: 1.4). The total number of cells was calculated in FIJI (NIH, USA) using the built-in Cell Counter plugin and the ratio of the live, apoptotic and necrotic cells were given as the % of the total cell number.