I'm planning to expose cancer cells to a certain concentration of a substance and am primarily looking to observe its mitotic activity.
Does anyone have any suggestions as to what cell assay I can use for this particular research? Or any related literature that could point me to the procedures?
In our lab we use the resazurin assay for measurement of cell viability and cell growth analysis. You can also do cell cycle analysis (e.g. propidium iodide).
Do you want to get a quick indication of metabolically active cells (viable cells)? If so it will be best to use a chromogenic assay such as the XTT or WST-1 assay (you can also use the older MTT assay but this is time consuming as you have to solubilise the colour). For the assay you initially incubate your cells with a concentration range of your substance (normally 24-72 hours). At the end of this you add the XTT or WST-1 substrate and incubate. Take absorbance readings every 30 minutes. Rate of absorbance change is indicative of cell number (i.e. mitosis). Results are then expressed as a % of the no substance conctrol.
Hi Do you want to monitor the number of cells after exposure? If so use the following:
Grow cells in a 96 well plate. At about 50 % confluence expose to a concentration range of your chemical. Incubate for 24-72 hours. Add XTT or WST-1 reagent (can be obtained from Sigma). Measure absorbance every 30 minutes. Change in absorbance is proportional to cell number. Express data as a % of your no chemical addition control. Will tell you if the chemical up or down-regulate mitosis. You can also buy the cell proliferation kits available from several other companies.
Yes i agree MTT or XTT are good for cell viability, and propidium iodide or anexin V are for cell cycle analysis. Also LDH measurements can be preformed to establish additional mechanisms. It depends what do you have to work with.
You can also use Sulforhodamine B colorimetric assay for cytotoxicity screening which is simpler faster and more sensitive than the MTT assay. It provides better linearity with cell number and is independent of intermediary metabolism.
http://www.nature.com/nprot/journal/v1/n3/abs/nprot.2006.179.html
Just go for cell cycle analysis by PI staining. Synchronize the cell at G0 phase by serum starvation and then release the cell. After that add your agent and give the cell time for minimum one complete cycle according to the doubling time of the cell. Assays the cell in S+G2/M in FACS analysis for mitotic level change.
Dear Jason, You can please refer to the link below to obtain certain valuable informations:-
http://www.lifeextension.com/Magazine/2010/4/Circulating-Tumor-Cell-Assays/Page-01
Moreover, you can also download the pdf attached herewith.
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