A cytotoxicity assay is incredibly useful in pharmacology and toxicology. In particular, it is used in an oncological research to rate compound toxicity, likewise a tumor cell growth inhibition during drug treatment. Those methods are rapid and inexpensive, however, the evaluation remains non-automatic and tedious for scientists.
It is feasible thanks to the different cells functions, e.g. changes in cell membrane potential, ATP production, co-enzyme production, enzyme activity and nucleotide uptake activity. Experiments in this area are focused on observing how many viable cells are remaining after the drug treatment , , .
One of the most commonly used parameter for this aim is the assessment of cell viability. It is ratio of live cells to all of them expressed as a percentage value , .
This application allows to make a quick, fully automated evaluation of live/death cells. It is dedicated for fluorescence staining with propidium iodide and AM calcein. The version for a trypan blue staining is under development. Those substances are the most frequently chosen dyes in the cytotoxicity assays .
In both staining methods, the state of cells membrane allows to distinguish between alive and dead cells.
How it is working?
In an environment with natural pH, the cell membrane is negatively charged. For this reason, anions are not transmitted through it. If the cell membrane is damaged, the voltage between the outer and the inner side of the cell will decline to 0. For this reason, anionic dyes will be able to delve into inside the cell , .
In the test with propidium iodide (PI) is being used the calcin AM dye simultaneously. PI marks dead cells by means of staining DNA red in the cell nucleus. On the other hand, AM delves into the cytoplasm and accumulates there in healthy cells, emitting light of green color , .
The cytoplasm in the test using Trypan blue remains clear for living cells with an undamaged cell membrane. In contrast, the cytoplasm for dead cells is blue-coloured .
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