I need the standard sequence assays step by step. I need a protocol for the study.
Merish,
As you mentioned, cell cycle profiling (to determine whether or not the cells respond to your treatment by arresting at one of the cell cycle stages) followed by apoptosis assay (using for example the Annexin V or TUNNEL assays) will be your next experiments. The protocol for these kinds of assays are straightforward and can be found in many related articles.
Once these assays are done, if you found an increased apoptosis and wanted to further focus, you can do a western blot analysis for the cleaved Caspase-3 to see if the final stages of late apoptosis have occurred.
Also you can determine whether the apoptosis of your cells is p53-, p21, or Rb-dependent by immunostaining.
To perform the anti-cancer drug assay on cancer cell lines, first determine the IC50 of drugs (concentration inhibiting growth of 50% cells) by conducting cytotoxic assay and determine the cell viability by Trypan Blue assay or MTT assay. Both are very common methods and protocols are available online. Once you have IC50 of your drugs, perform cell cycle analysis (Propidium iodide staining) and apoptosis assay (Annexin V) using IC50 concentration to determine the mechanism of cell death. You can also check for the expression of cell cycle associated proteins, apoptotic proteins and other pro-survival pathways like AKT/NF-kB. Once you have in vitro data, you can go for in vivo studies to validate your in vitro results.
Dear Sandeep,
Can you please let us know how the in vivo experiments should be designed? What should be investigated?
You can use human xenograft orthotopic mouse model to study efficacy of anti-tumor drugs. Implant tumor cell lines to SCID or Nude mice. Let them grow for 2-3 weeks and when tumor sizes reaches 150mm3, start treatment with different anti-cancer drugs with selected doses for 1-2 weeks. Keep measuring tumor growth every 2 days for a period of 1-2 month. If drugs are effective you will see a significant difference in tumor growth in untreated and treated group..
And what about the different growth rate and metastasis ability of different cancer cells? Due to their rapid growth, some tumors would lead to death in shorter period of time than what you suggested.
You will be testing one cell line at one time and one drug cannot be used against all cancer cells. The in vitro data will give u estimation abut which drug might give u significant results in in vivo settings.
Dear, Pls kindly after MTT, go to mophological detection either by Ao-PI, or Hos chest test, then flowcytometry for annexin, cell cycle, and tunel. Then SEM and TEM and lastly Caspase protease activity. You can also try western blot and PCR. Pls kindly look at attached files. TQ
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