I would like to add that cell proliferation results in increase in cell number. Therefore, depending upon the cell type, one can use various approaches to count the actual number of cells that are healthy in a given time, which is appropriate for increase in cell number with or without replenishing cell growth/culture medium.
If you can have a good support from the flow cytometry facility at your institution, a flow cytometry-based method using "CellTrace" kits from Invitrogen (Life Technologies) could be worth considering.
A colony formation (clonogenic) assay might be worth considering if you have multiple treatments / cell lines you are wanting to compare.
Or if you have access to FACs you could halt your cells in G1 (I find hydroxyurea overnight is easiest) and then wash out and take samples every 2h to see how fast the cells are progressing through the cell cycle.
Before anything I'd try counting cells on Neubawer and Tripan Blue staining, along time (6, 12, 24, 48, 72, Hs) until plateau at different seedding cell density before using floy cytometry or MTT. Find conditions for detecting treatment influences on cell growing and then corroborate with more complex techniques.
Thanks a lot to everybody for your wonderful suggestions. yes, we have flow cytometry available in our institute.
There are various methods (several examples provided below) to evaluate cellular proliferation but at different endpoints.
[1] A simplest way is to directly count cell numbers at different days after plating.
[2] A colorimetric assay based on MTT, XTT, MTS, WST-1 or WST-1 measures the enzymatic activity of mitochondrial dehydrogenase (succinate-tetrazolium reductase) that converts these tetrazolium salts into purple colored insoluble formazan. A similar assay employing resazurin or AlamarBlue can be used both colorimetrically and fluorometrically. The assay of this type evaluates the mitochondrial metabolism.
[3] Tritiated thymidine uses radioisotope and evaluates the DNA synthesis. A similar assay employing other thymidine analogues BrdU, IdU, CldU or EdU does not need the use of radioisotope. There have been S phase markers available, such as Ki-67, PCNA, DNA topoisomerase II, and histone H3. Evaluation of E2F (required for a transition from G1 to S) or other proliferation-related markers may also be useful.
[4] A dye exclusion assay measures the membrane permeability and uses colorimetric (e.g., trypan blue, Erythrosin B) or fluorescent dyes (e.g., propidium iodide, ethydium bromide).
[5] A colony formation or clonogenic assay measures the ability of single cells to form colonies with 50 or more cells, usually in about two weeks after plating.
If there is difference in cell proliferation, then flow cytometry may be conducted, followed by analyses of molecular changes in relation to cell cycle checkpoints.
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