JoVE Encyclopedia of Experiments
Cancer Research
0 views • 4:17 min • April 30th, 2023
- Tyrosine Kinase Inhibitors, TKI, blocks the kinase receptor from binding to ATP, preventing the phosphorylation. Begin with plating cancer cells in growth media. Allow them to reach favorable density, and transfer the cells to multiple wells. After overnight growth, dispense different drug concentrations in multiple wells and let the cells grow for the desired duration. Add MTT, a water soluble dye, to measure cell viability. Mitochondrial enzymes convert MTT to insoluble purple formazan inside living cells.
Stop the reaction by adding a suitable solubilizing agent and measure formazan spectrophotometrically. This method is called the MTT assay. Plot a graph of living cells indicated by formazan against drug concentrations. Calculate inhibitory concentration, IC50, by finding drug concentration that kills 50% of cells using the software.
Next, grow cancer cells in the growth medium and distribute them in three different plates. After the cells reach subconfluency add 1/10 dilution of IC50 concentration. Allow the cells to grow and perform the MTT assay. Repeat this process with increasing drug dosage. Cancerous cells slowly develop resistance to TKIs due to mutations in the prote
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