Celltiter 96 non radioactive mtt assay kit

For multiple timepoints, a total number of 2 × 10⁴ GP33-specific CD8+ T_eff and T_mem or both were separately co-cultured with 2 × 10⁴ B16GP33 melanoma cells at an overall effector:target ratio of 1:1 in 48-well plates in the presence of 10 ng/mL recombinant human IL-2 (BD Biosciences, San Jose, CA) for MTT assays as previously described [7 (link)]. T cells were characterized and isolated using the methodology described for adoptive cell transfer. Viable B16GP33 melanoma cells were quantified using the Celltiter 96 non-radioactive MTT assay kit (Promega, Madison, WI) following the manufacturer’s instructions.

The SCC25, SCC104 and HPEK cells (1.8 × 10⁴/ well in 200 μl culture medium in a 96-well plate) were treated with different doses (1, 10, 100, 500, 1000 and 5000 nM) of TM-025 and TM-026 for 24, 48 and 72 h. Percentage viability of cells was evaluated by the MTT assay, a putative test that evaluates formazan formation [32 (link),33 (link)], for computation of mitochondrial succinate dehydrogenase activity as an estimate of live cells [34 (link)]. The experiment was performed using the Cell Titer 96 Non-Radioactive MTT assay kit (Promega Biosystems, CA), according to the manufacturer’s instructions. Cells treated with DMSO (1 μl per 200 μl medium) were considered as control groups (vehicle). End-point absorbance was determined at 570 nm (with a 650 nm reference) using a Synergy HT Multi-Mode Microplate Reader (BioTek U.S., VT). The percentage viability of cells were calculated estimated using the following formula: % viability = (A_T/ A_V) × 100, where A_V is the absorbance of the vehicle-treated control group and A_T is the absorbance of the test samples (TM-025/TM-026-treated).