Question

Design a series of experiments to test whether a chemical drug induces cell death through intrinsic apoptotic pathway.

Please help ASAP!

Answer 1

First MTT assay is performed to check the cell death or toxicity potential of a product. If cell death occur and you want to check weather it is especially occuring via intrinsic pathway of aoptosis or not then do the following experiments

1. Western blot in whole cell lysate after drug treatment: Check the expression of BAX and BID which will increase on the drug treatment in compare to control.

2. Westren blot of Mitochondrial lysate: In intrinsic apoptotic pathway the permeabilisation of the mitochondria occurs resulting in the release of cytochrome c to the cytosol. So after isolating the mitochondia, cheak the expression of cytochrome c protein. If the expression of cytochrome c is lower in the drug treated mitochondia then the drug is inducing intrinsic apoptosis,

3. DNA fragmentation assay: In intrinsic apoptosis DNA fragmentaion occurs. So if the chemical drug induces intrinsic apoptosis then more fragments of DNA will result.

4. Microscopic analysis: Transmission electron microscopic analysis of mitochondrial structure.

5. Flowcytometry: JC-1 dye staining for Mitochondrial Membrane Potential by flowcytometry. Accumulation of mitochondria, will be indicated by a fluorescence emission shift from green color (~529 nm) to red (~590 nm). In other words, mitochondrial depolarization will be indicated by a decrease in the red/green fluorescence intensity ratio.