I want to conduct a proliferation assay to test a chemotherapy combination on a cancer cell line. At first, I thought about the MTT assay. But now, all sources are saying that these do not measure proliferation.
If you had a hypothesis that some drugs decrease proliferation, how would you go on about it? what experiments/assays would you use?
Hello, Aicha!
1. Probably the simplest analysis for proliferation is to sow the same number of cells, treat with drugs and after the same time count the number of cells (for example, in a Goryaev chamber or using an automatic cell counter). Then, in relation to the ratio of the grown cells to the initial number, the proliferation index in% can be estimated.
2. You can use antibodies to marers of proliferation Ki-67, PCNA and use flow cytometry or immunofluorescence.
3. MTT test can also be used. The viability of cancer cells will be proportional to their proliferation. If your drugs act on proliferation, then cell viability will also change.
Indeed MTT assay measures cell viability and not cell proliferation. There are a few methods to measure cell proliferation: https://www.labome.com/method/Cell-Proliferation-Assays-and-Cell-Viability-Assays.html
Counting cells could be a very easy way. MTT can also be used over different time points. Yet, the results should consider effects of drugs on metabolism.
Hello, Alexandr Chernov
Thank you so much, that was very insightful!
There is only one reliable way to measure proliferation (assuming you define this as increased cell number), and that is manual cell counting!
All other methods mentioned above are indirect, or accompanied by large errors/assumptions.
Buy a cell counting chamber (Hemocyometer, ~US$100) and use the microscope. It takes just minutes, and also delivers you a bonus – confidence and peace of mind
Cell proliferation can be measured either by counting the cells after treating them with your compounds using trypan blue dye in hemocytometer under microscope or by incorporating/staining cells with CFSE/eFluor/Ki67 dyea and check through flow cytometer.
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