Variskan flash plate reader

Enzyme activity assays for cathepsin B (cat# ab65300, Abcam), cathepsin D (cat# ab65302, Abcam), and cathepsin G (cat# ab126780, Abcam) were performed on three snap-frozen MDOTSCC samples of the cohort of six patients used for NanoString mRNA analysis, according to the manufacturer’s protocol. A snap-frozen tonsil sample was used as an appropriate positive control for the cathepsins B (17 (link)) and D (18 (link)) assays, and a denatured sample of the same tonsil was used as an appropriate negative control. The capthepsin G assay kit was supplied with its positive and negative controls. The results were obtained using the Variskan Flash plate reader (Thermo Fisher Scientific). Experiments were performed in duplicates with averages taken for each.

Cell lines were seeded in 96-well plates at a density of 3 × 10³ cells/well and treated with a range of CX-5461 concentrations for 96 h and cell viability was measured using [3-(4,5-dimethylthiazol-2-yl)-5-3(carboxymethoxyphenyl0-2-(4-sulfophenyl)-2H-tetrazolium] (MTS) assay according to the manufacturers protocol. A quantity of 100 μL of Cell Titre 96 Aqueous Non-Radioactive Cell Proliferation (MTS) Assay (Promega, Southampton, UK) was then diluted to 1:10 using RPMI 1640 media and added to each well, including empty wells for use as a background control. Optical density was measured using a Variskan Flash plate reader (Thermo Scientific, Altrincham, UK) at 492 nm wavelength. Each MTS assay was performed in triplicate and repeated at least three times. For clonogenic assays, cells were grown in 6-well plates and following treatment, incubated for up to 14 days. Colonies were fixed in 50% ethanol in PBS and stained with 5% crystal violet in PBS (Sigma Aldrich, HaverHill, UK). Colonies with more than 50 cells were counted and survival fractions were determined taking into consideration the plating efficiency for all treatment modalities based on triplicate experiments. Calculations were based on established protocols.