I have expressed some genes in leukemic cells lines, which are previously not present in those cell lines. I would like to know if these genes confer any proliferative advantage. I am doing conventional cell counting expt spread over 10 days and i was thinking of using MTT assay as an additional tool to know the effect of these genes on proliferation.
Has anybody used MTT assay to study the proliferation kinetics? Thanks in advance.
MTT is not only dependent on the cell number but also on the metabolism of the cell (NADH/NADPH production). Do you know if your genes should also effect the glycolysis or respiration of our cells? Because than you would see a change in MTT even if there is no change in proliferation rates between your conditions.
You could also think about doing BrdU or 3H-Thymidin assay, which tell you how many cells are entering the S-phase of the cell cycle. However, entering the cell cycle does not mean the cells are fully dividing (they can still arrest in eg M-phase)
I did a lot of different inhibitor experiments with my cells and found that the most reliable method for seeing proliferation differences over time is (as you already did) is counting the cell number. I'm sorry....
Nevertheless, you can think about a combination of all three methods to clearify why your cells have a growth advantage (metabolism vs. cell cycle effects)
Best wishes
MTT is not only dependent on the cell number but also on the metabolism of the cell (NADH/NADPH production). Do you know if your genes should also effect the glycolysis or respiration of our cells? Because than you would see a change in MTT even if there is no change in proliferation rates between your conditions.
You could also think about doing BrdU or 3H-Thymidin assay, which tell you how many cells are entering the S-phase of the cell cycle. However, entering the cell cycle does not mean the cells are fully dividing (they can still arrest in eg M-phase)
I did a lot of different inhibitor experiments with my cells and found that the most reliable method for seeing proliferation differences over time is (as you already did) is counting the cell number. I'm sorry....
Nevertheless, you can think about a combination of all three methods to clearify why your cells have a growth advantage (metabolism vs. cell cycle effects)
Best wishes
Noorjahan,
As has previously been discussed with in the above answers which all bring up some very valid points, I might suggest that you conduct a dye dilution experiment in which cells are labeled with a given dye by incubation and then using flow cytometry the absorbance as a result of the concentration of the dye is measured. The principle underlying this is that each time a cell divides each of the daughter cell receives about half of the dye from the parental cell and this is then diluted as the cell grows (in terms of volume). doing this experiment will show you the populations of cells that have divided and thus proportion of cells that have divided and provide insight into how many times each counted cell has divided since labeling the cell (again based on the concentration of dye inside each cell). Generally, you can assess three to four generations of cells this way before the dye becomes too dilute.
I hope this helps.
Andrew
Thanks Florian, Maren and Andrew. At present i am combining three different approaches. Conventional cell counting, MTT assay and also CFSE (dye based dilution assay). With these assays, I hope to get an idea of the functional of the gene I am studying.
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