there are many agents approved to have anticancer agent using MTT or any other type of tests but yet the mechanism of how they induce anti proliferation or apoptotic activity is not reveled, So how to determine their mechanism of action .
Hi Wafa Elamin, you can perform several flow cytometry assays, such as cell cycle (using PI), apoptosis (using Annexing V/PI), autophagy (using LC3) or even differentiation markers in order to correlate the anti proliferative effect of an antitumoral agent.
Could always try using a C13 labelled version and cross linking it with its targets using formaldehyde before isolating with SDS page and characterizing the targets using mass spectroscopy.
Use radio labeled moieties to interdependently follow the inhibition kinetics of DNA, RNA and protein synthesis. Following O2 or glucose consumption, can be used as a measure of the effects on energy metabolism etc. These are easy first approaches, that could give you some clues.
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