Could anyone suggest techniques for testing the following 2 Hallmarks of Cancer: 1. Resisting Cell Death & 2. Activating Invasion and metastasis? Any literature on this would be appreciated as well. Thanks
Resisting cell death
1. PI-FACS for DNA fragmentation
Resisting cell death after PARP cleavage
1. Blebbishield mediated sphere formation with drug of your choice
LiCl+Cisplatin is done in my paper
https://www.researchgate.net/publication/233742776_Blebbishields_the_Emergency_Program_for_Cancer_Stem_Cells_Sphere_Formation_and_Tumorigenesis_after_Apoptosis
Invasion and metastasis
1.Scratch assay
2.Transwell migration assay
3. Metastasis in mice model of your choice either by orthotopic or ectopic or tail vein route.
Good luck.
Article Blebbishields, the Emergency Program for Cancer Stem Cells: ...
for anti-apoptosis, you can also investigate caspase 3/7 activity by using commercial kit from Millipore.. FACS or reading absorption. depend on your perference..
This somewhat depends on your sample/material. What do you plan on testing? My guess would be tumor cell lines in cell culture? If you let me know I'll be glad to give my 2 cents on which assays to use.
Simply you can test:
1- Motility or migration assay using the Boyden chamber .
2- Invasion asay using the matrigel invasion chamber.
3- Adhesion assay.
4- Colony forming assay.
it depend on your sample, is it blood? or FFPE or any solution please specify
Hi Nicholas Wagner, you are correct, I am using s squamous cell carcinoma cell line. Thanks!!
Hi there!
For resistance, we would normally do a basic proliferation assay (MTT or MTS), with and without a drug treatment. You can also use more molecular techniques (such as PARP cleavage) if you know the drug should cause apoptosis.
As for Q2......
There is a really nice cell motility kit you can get from Thermo Scientific, (#K0800011). Nice pictures, you can do it in a 96 well plate, and much better than the traditional scratch assay systems. See the following publication (Figure 1) ....(attached)
All the best
Hi Alexis,
Question 1:
Éamon is absolutely right. Typically you would measure first basic proliferation using an "easy" assay such as MTT or WST-1 (or measure cell death, e.g. using crystal violet).
If you see effects, you will likely want to go more into detail to pinpoint which mechanism is causing cell death. Here the first step is usually the discrimination between apoptosis and necrosis. Apoptosis is most commonly detected with either Annexin-V (binds to Phosphatidylserin, which is transported to outer membrane during apoptosis) or by detection of the caspase cascade, in which PARP is cleaved by caspases. Necrosis can be tested by nucleic acid probes, which are impermeant to live or apoptotic cells, but stain necrotic cells (such as propidium iodide or EthD-III).
I have to pass on quesiton 2, but I think Éamon is leading you onto the right track there as well :-)
Good luck!
Nick
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