Im currently working on SH-SY5Y cell line. The treated group showed increased BrdU +ve cells on Immunocytochemistry technique but % cell viability from MTT assay showed no significant effect. If there's increase in cell proliferation, % cell viability should also increase, at the same time period of treatment. I would really appreciate your help analyzing this case. Thank you.
Hi Rubina,
BrdU will only incorporate in cells that are newly synthesizing DNA at the time that you treated the cells.
An MTT assay will assess the number of viable cells at a given time point. The colorimetric assay is depended on the cell metabolic activity, which is suppose to correlate with increase in cell numbers. Depending on the action of your experimental compound you may have to consider other confounding variables such as disruption of cell metabolic activity.
Chan el at 2013 "Therefore, differences in compound mechanism of action and cell line-specific responses can yield significantly misleading results when using ATP or tetrazolium-reduction assays as a proxy for cell number when screening compounds for antiproliferative activity or profiling panels of cell lines for drug sensitivity."
See article below:
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3656927/
Hi Rubina,
BrdU will only incorporate in cells that are newly synthesizing DNA at the time that you treated the cells.
An MTT assay will assess the number of viable cells at a given time point. The colorimetric assay is depended on the cell metabolic activity, which is suppose to correlate with increase in cell numbers. Depending on the action of your experimental compound you may have to consider other confounding variables such as disruption of cell metabolic activity.
Chan el at 2013 "Therefore, differences in compound mechanism of action and cell line-specific responses can yield significantly misleading results when using ATP or tetrazolium-reduction assays as a proxy for cell number when screening compounds for antiproliferative activity or profiling panels of cell lines for drug sensitivity."
See article below:
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3656927/
BrdU labeling time should be short, e.g., 2-4 hours and not longer than 24 h. If this condition is met, try Cell Proliferation Reagent WST-1, because WST-1 does not need enter cells while MTT needs entering cells and the reduced production is exocytosed.
http://www.sigmaaldrich.com/catalog/product/roche/cellproro?lang=en®ion=US
I would not compare MTT with BrdU. There are a lot of " if" to consider.
Best
I will say that MTT assay is rather qualitative than quantitative, as it does not give you an exact percentage of viable cells. Cell proliferation does not mean cell viability, as it is possible that some cells die after they divide, but these dead cells still retain BrdU in them. I would suggest to use a fixable viability stain and check using flow cytometry for exact viability, if knowing viability rate is really important for you.
The protocol depends on what are you interested on studding. If you want to studdy cell proliferation, ensure cells are at the growinfg fase of the growing curve. But if you are studing cell viability or apoptosis, ensure your treatment is on plateau. One suitable thechique to study both aspects together is staining fixed cells with DAPI or IP/RNasa and analize DNA content by flow citometry. MTT assay allwais gives confusing results when mitosis apoptosis and necrosis are present at the same time.
http://www.abcam.com/protocols/flow-cytometric-analysis-of-cell-cycle-with-propidium-iodide-dna-staining
Hi, please check out https://www.researchgate.net/post/Can_I_use_the_MTT_assay_for_cell_proliferation/1
Hope this helps
The MTT readout is a measure of total metabolic activity in a cell culture. This is a mixture of cell proliferation rate, cell size, metabolic rate and cell survival. If you are fairly sure that three of these four factors are not differing much between the treatments or times you are comparing, then it will give you a measure of the fourth one. So MTT will be a good measure of proliferation ONLY if death rate, metabolic rate and cell size are not differing noticeably between treatments (or times). It will be a measure of cell survival if the cells are not proliferating or changing metabolic rate or size. Etc.
Often this kind of test is not very useful, or only as a first look (quick and dirty, as everyone says). Needs validation by another assay of the parameter you actually want to measure.
Please check the question and related answers :
Can % viability be considered an accurate representation for proliferation measure by MTT assay?
Hope it would be useful.
I am in complete agreement with the valuable answer of Professor Dorothy C Bennett.
Dr Bennett's is the comprehensive answer. Use instead a particle cell counter (Couldter Counter or alike).
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