Melanoma Cell Lab Report

To verify whether the melanoma cells are able to survive and form colonies in vitro starting from one single cell in presence of BTK inhibitors a colony assay was performed. This assay can determine the efficacy of these specific agents. A very diluted amount of cells, 1.500 cells/well, were seeded in duplicate in 6 –well transparent plates (Euroclone®) in 2 ml of medium. The next days, when the cells are attached to the bottom of the wells, the cells were treated with the BTK inhibitors ibrutinib, AVL 292 and RN-486. The concentrations used were the same as for the growth curve experiment. Every 48 or 72h the cells received fresh medium containing the same concentrations of BTK inhibitors. After checking the colonies with the microscope, this step was repeated till the colonies reached a certain confluence. When this point was reached, the medium was discarded, the plates were washed with PBS and the colonies were coloured and fixed with the crystal violet solution containing 35% of ethanol. Subsequently, the next day the dried plates were photographed with Gbox iChemi imaging system (Syngene®). …show more content…
Toxicity assay
For this experiment 20.000 cells/well were seeded in 96-well transparent plates (Euroclone®) and incubated overnight. With this number of cells, a 50 to 60% confluence should be obtained, an optimal range to shown all the possible drug effects: cytotoxic, anti-proliferative (cytostatic) or no suppressing effect. The next morning, cell attachment was checked by microscope, before the treatments were prepared by diluting the initials solutions. The drugs and the concentrations (derived from literature) used for the experiments were:
Table 3.2: Overview of the used drugs, their function and the used

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