Gaphpad prism v 5

Cell viability was evaluated by the tetrazolium dye (MTS) Short-Term Cytotoxicity Assay. In brief, cells were seeded in 96 well plates at 5,000 cells/well. Twenty-four hours after cell plating, media was removed and replaced with fresh media containing test compounds IQ3A, TMPyP4 and 5-FU, or vehicle control (DMSO). Following 72 h of compound exposure, cell viability was evaluated using the CellTiter 96 AQueous Non-Radioactive Cell Proliferation Assay (Promega, Madison, WI, USA), using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) as previously described [34 (link)] [35 (link)]. Cell viability data were expressed as mean ±SD from at least three independent experiments. IC₅₀ and IC₆₅ values were determined using GaphPad Prism v.5.00 (GraphPad Software). In selected experiments, cell viability was also assessed by trypan blue exclusion assay [34 (link)], and general cell death by LDH activity from cell culture supernatants [7 (link)].

HCT116 human colon carcinoma cells, SW620 human colorectal adenocarcinoma and HEK293 T human embryonic kidney cells were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, and 1% antibiotic/antimycotic (Invitrogen, Grand Island, NY, USA) and maintained at 37°C in a humidified atmosphere of 5% CO₂. Cells were seeded in 96 well plates at 5.000 cells/well. Twenty-four hours after cell plating, media was removed and replaced with fresh media containing test compounds and 5-FU (Sigma), a common cytotoxic agent used in colon cancer treatmen8 (link)9 (link)41 (link), or vehicle control (DMSO). Following 72 h of compound exposure, cell viability was evaluated using the CellTiter 96 AQueous Non-Radioactive Cell Proliferation Assay (Promega, Madison, WI, USA), using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) as previously described42 (link). Cell viability data were expressed as mean ± SEM or mean ± SD from at least three independent experiments. IC₅₀ and IC₆₅ values were determined using GaphPad Prism v.5.00 (GraphPad Software). (Performed at Cell Function and Therapeutic Targeting Group, iMed.ULisboa)