Hello,
My lab is testing the % surviving cell rate(neuroblastoma SH SY5Y) under 6-ODHA. By doing literature search, we realized that people tend to use MTT assay for determine % surviving cell rate. Since our lab only has LIVE/DEAD® Viability, we are deciding do we need to buy MTT assay instead.
Do you guys think we should use MTT but not live/dead assay? Why MTT seems more popular than live/dead assay?
Any input will be appreciated =]
The short answer is no. There are many different assays that are used to determine cell survival/death. MTT assay, like LIVE/DEAD® Viability assay, is one e of many cell viability assays used to check cells response to drugs etc. They tell whether a cell survives or dies in response to the treatment, however most of these assays fail to tell what mode of cell death is used. As such after using a cell viability assay other methods need to be used to determine the mode of cell death (if that is required). Please see this paper in which we used 6-ODHA in PC12 cells. We used several assays including MTT.
http://citeseerx.ist.psu.edu/viewdoc/download?doi=10.1.1.326.8902&rep=rep1&type=pdf
MTT is (typically) cheaper than live/dead assay. Also, it doesn't really tell you if any cells have died. It measures the amount of living cells based on how many living mitochondia are present.
If you are interested in cell death (or how they died) then, as Afshin says, other methods should be used. If you are only interested in growth response, MTT should suffice.
You mention you wish to test the % survival rate. Either the Live/Dead Viability Assay or the MTT will work there. MTT is of course cheaper. If you aim to present the number of dead cells too, Live/Dead Assay will work. If you are interested on the type of cell death, then you need specific assays, such as Annexing V assay for apoptosis. You may have to use more than one type of assay in such case, as the other commenters here emphasized.
We used both Live/Dead Viability Assay (Fluorescent-microscopy based), and MTT assay in our lab. Live/Dead Assay worked well in viral transduction assays, whereas MTT gave us idea on the survival rate of our cell lines against different agents, as a preliminary test for further more specific tests. We perform 6 repeats in MTT tests, as it is based on spectrophotometric reading, which may fluctuate due to a number of factors.
Wishing you great results :)
Are you using proliferating cultures and treatment time of more than ~4 h? Then please read this. Viability assessment with proliferating cultures requires single cell analysis and cannot be done in a 96-plate format using a plate reader. I wrote the following section for the most misleading crystal violet "cytotoxicity" assay which applies to all 96-well plate "viability" assays. A major advance in cancer research will be made when such misinforming and useless assays will be removed. "The Crystal Violet "Cytotoxicity" assay with proliferating cultures will determine anything but cytotoxicity. The 2016 protocol description by Feokistova et al. is for non-proliferating cells. Please see their Fig. 1 (control wells before and after treatment; there is no change in cell number!!!). In fact, the description of all such 96-well plate assays (MTT, CellTitre-Glo, etc) for viability assessment with proliferating cultures are majorly flawed. Misinformation perpetuated by misuse of such assays, as well as large-dye uptake assays (trypan blue, PI) have been discussed (PMID 30071623 and refs therein, including PMID: 27750219). If you use the single-cell MTT assay described in PMID 30071623, you will see that cells that are assumed to be dead (even with apoptotic morphology) are viable and metabolize MTT. Like the "gold standard" colony formation assay, all 96-well plate "viability" or "cytotoxicity" assays that are performed with proliferating cultures determine the ability of a test agent to convert proliferating (dangerous) cancer cells to dormant (most dangerous, tumor repopulating) cancer cells, and not to dead cancer cells. The IC50 values measured by such assays reflects a sum of cell cycle checkpoint activation (a pro-survival mechanism), short-term dormancy due to reversible apoptosis (anastasis), and early stages of durable proliferation arrest (dormancy, due to polyploidy, multinucleation and senescence, all of which contribute to disease relapse). None of these well-documented responses is associated with loss of viability. Viability assessment with proliferating cultures requires single cell assays and cannot be determined in a multiwell plate format using a plate reader."
Razmik Mirzayans do you happen to have a protocol for a single cell analysis approach to cell viability assessment?
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