Celltitre glo 3 d luminescent assay

Cells were plated at 4 × 10⁴ cells per well into 96-well plates and incubated for 24 h before media were replaced with test media (0 h) supplemented with glucose (5.6 mM), glutamine (2 mM), 10% FBS, lactic acid (0–20 mM) and uprosertib (5 or 10 μM) or vehicle (0.1% DMSO). Apoptosis was determined at 24 or 48 h post initial treatment using the Caspase-Glo 3/7 assay system according to the manufacturer’s instructions (Promega, Madison, Wisconsin, US). ATP was measured after 24 h of treatment using the CellTitre-Glo® 3-D luminescent assay (Promega, Madison, Wisconsin, US) according to the manufacturer’s instructions. Luminescence was measured using the CLARIOstar plate reader (BMG Labtech, Ortenberg, Germany) and readings were normalised first to cell density determined using an SRB assay performed in a parallel plate and second to the vehicle controls.

HCT116 cells were plated into 96-well Ultra-Low Attachment plates (Corning, New York, US) at a density of 1 × 10³ cells per well in 50 μL of media supplemented with glucose (5.6 mM), glutamine (2 mM), 10% FBS and lactic acid (0 or 10 mM) and incubated for 24 h to allow 3-D spheroids to form. Subsequently, 50 μL of test media containing uprosertib (0 to 15 μM) was added on top of the original 50 μL and spheroids were incubated for 72 h. Brightfield images of spheroids were obtained using the IN Cell Analyzer 2000 (GE Healthcare, Chicago, Illinois, US) using a 4 × 0.2 NA objective lens. Scale bars represent 100 μm and were added using Image-J software. To quantify spheroid viability after 72 h of uprosertib treatment, we used the CellTitre-Glo® 3-D luminescent assay (Promega, Madison, Wisconsin, US) according to the manufacturer’s instructions.