Celltiter 96 non radioactive cell proliferation assay mts

Female 5-week-old NMRI-Foxn1 nu/nu mice (16–20 g) (Janvier Labs, France) were injected subcutaneously with 4 × 10⁶ of SKMEL28 cells or, after Ketamine/Xylazine anesthesia, were xenografted with a patient-derived BRAF V600E-mutated tumor tissue in the brown fat. Mice were euthanized by cervical dislocation when their tumor reached a volume of approximately 1-1.5 cm³. Tumors were excised and a representative tumor piece was washed with PBS and sliced. Tumor sections were then placed in wells coated with gelatin (Sigma-Aldrich, USA) in 24-well plates in quadruplicates. The tumor sections were incubated for 8 days at 37 °C in 250 μL complete media supplemented with 10 μM dabrafenib (GSK2118436, Selleckchem), 1 μM cobimetinib (GDC-0973, RG7420, Selleckchem), or a combination of both inhibitors dissolved in DMSO. As controls, some wells received an equivalent volume of medium containing 0.5% DMSO. The medium was replenished every two days, followed by a day off of treatment for the intermittent dosing schedule. At the end of the incubation period, cell proliferation was measured using the CellTiter 96® nonradioactive cell proliferation assay (MTS) (Promega, France), according to the manufacturer's instructions. HDRAs were conducted in 3 independent experiments.

For cell proliferation assays, cells were seeded in 96-well plates at 2×10³ cells/well at a final volume of 100 µL and incubated overnight. The viability of cells was determined with a CellTiter 96 non-radioactive cell proliferation assay (MTS) (Promega BioSciences, USA) following the manufacturer’s protocol.