Self-made Alamar Blue (cheap and quite sensitive if you seed the right amounts of cells and optimize detection times for each cell type). However, recently I found out that Quant-iT™ PicoGreen® dsDNA Assay measuring DNA content (thus number of proliferating cells) gives different results than AB metabolic activity assay.
Anyone interested in this topic should read the publication "Discrepancies between metabolic acitivty and DNA content as tool to assess cell proliferation in cancer research" by Quent el al. (see attached)
In addition, please see my website and learn how to make your own AlamarBlue in order to stop paying more than you have to.
http://www.cellsignet.com/protocols/buffers.html
I have used just some of them and will not be able to say which one is he best. But, I do know each assay method is based upon certain cell metabolism. Some researchers, especially when you submit your manuscript and receive the criticism from the reviewers, may insist one is far better than the others but I do have the doubt. At least I know XTT/MTT you need to have apppropriate mitochondrail functions. But when you compare two cell cultures you are not sure your treatment will affect the mitochondria count in the cell or not. Trypan blue can kill the cells if you let it sit long enough. Hemocytometer manual counting maybe more reaonable but will take a long time which may lead to several hours difference when you count different containers. Also, people sometimes ignore those cells already detached which may have large quantity than you can imaging. I always think at least two assay using different methods with similar finding will be more reasonable.
Dear Dr. Sathishkumar D. Tala
Alamar blue is cheaper and much easier and safer to use with cell culture studies; The choice of selecting between formazone based detection it purely depended on cells metabolic rate and site of metabolic action, even assay conditions can alter metabolic activity and sometime tetarazoliuim dyes seems to be toxic to few cell lines even at lowest concentration of 50 µg/mL. Hence while performing cell viability with different tetarazolium dyes they produce different results depending on whether they are reduced intracellularly (MTT, MTS) or extracellularly (WST-1). You have to consider few more important things like, while Alamar blue quantification with fluorescence check for background signal from other compounds. And with MTT, there are many reports available pretending that MTT can be cross reactive with many compounds (like Ascorbic acid) hence there are many chance for MTT to get reduced and shows false positive results. I agree with Ru-Jeng Teng statement on Trypan blue which upon exposing for longer time; it will definitely kills your cells.
All the best
If you are interested in metabolism, or using it as a readout for reduced cell activity in response to a compound/treatment condition then MTT is a very good place to start. However, if you are more interested in proliferation of the cells rather than activity then a dye & flow cytometry based assay such as CFSE is very useful.
@ Babak Beikzadeh
i am looking for cancer cells like solid tumor and leukemia
But what exactly do you want to measure? Cytoxicity, viability, cell proliferation?
thanks to all,
We used to measure IC50 value for our synthesized compounds with Alamar Blue assay, and i want to know about other assay.
We use WST-1 to measure cell survival to various types of treatments in different cancer cell lines with good results and reproducibility. Furthermore, it's quick and easy.
depends. Im familiar with alamarblue. one good thing is that with some experiemtnal parameters, you could use the same experimental plate for each time point.
However, it has its drawbacks, you coudl have a couple of highly metabolic cells on a plate where there is little proliferation and the opposite in both cases in another treatment. always best to perform proliferative assay (picoGreen, live/dead assay in combination
MTT works simple, so it is great when you want to save time and money.
We used MTT for IC50 but could not get consistent result then we used cell titre blue (fluorescence) which was perfect. MTT doesn't work efficiently with any compound with high antioxidant properties.
ALamar blue without any restriction... For cell proliferation of course...
MTT Assay is best to do in invitro Cytotoxic study as Most research work done to check the viable and non-viable cells, is done by this method and You will get many research papers on it and number of good protocols.
We have compared MTT, AlamarBlue and PrestoBlue for viability assays and we obtained the same results in %viablity, so I think they are all reliable. The good thing about AlamarBlue or PrestoBlue is that you can perform the analysis at different time points in the same plate, and you can use those cells later, since it is not toxic at all.
We use three different assays, MTT, SRB and CVE. Each one has adnantages and disantvantages. By comparing both of them you will have a reliable results. I can send you a paper of our team with each assay. It depends on the cells, the drug you want to use etc.
Self-made Alamar Blue (cheap and quite sensitive if you seed the right amounts of cells and optimize detection times for each cell type). However, recently I found out that Quant-iT™ PicoGreen® dsDNA Assay measuring DNA content (thus number of proliferating cells) gives different results than AB metabolic activity assay.
Anyone interested in this topic should read the publication "Discrepancies between metabolic acitivty and DNA content as tool to assess cell proliferation in cancer research" by Quent el al. (see attached)
In addition, please see my website and learn how to make your own AlamarBlue in order to stop paying more than you have to.
http://www.cellsignet.com/protocols/buffers.html
You could also try to use a WST-1 assay, which detects the capacity of the mitochondria of viable cells to convert WST-1 reagent (Roche), WST-1 cell viability/cytotoxicity assays might relatively be most easy to perform.
I think that it depends on your application. For example, our lab studies doxorubicin and related variants, which are known to have effects on the mitochondria. Therefore for cytotoxicity measures, I tend to avoid assays that require mitochondrial activity, because perhaps there are sublethal effects of the drugs on the mitochondria that would contribute to an difference in readout between my experimentals and my untreated controls.
I completely agree with Ben Barthel. All assays that involve redox reactions such as the XTT/MTT/WST, and the ATP assays, are potentially affected by events that are not immediately connected to cytotoxicity. And, as Ben says, if you are working with an agent that can be suspected to in one way or another affect mitochondria or metabolism (without actually killing), you risk getting a quite false impression of high cytotoxicity. With doses of cisplatin that don´t really induce any death within 10-15 hours, I have seen substantial dips in MTT values at timepoints around 2-4 hours, i.e., after 6-7 h the values are back to baseline. Moreover, I object to these assays being called cytotoxicity assays - they are viability assays. The question they usually answer is "What is the proportion of viable cells in this well, compared to untreated wells?"
This is an interesting discussion that has been going on for years. We used to use radioactive materials based assays. I have not used all the assays discussed. We use various live/dead stains, clone expansion assays, MTT, Trypan Blue, and LDH to support our primary in house viability assay which is the alamar blue (rezasurin) assay. We selected the alamar blue assay as our primary assay for reasons well articulated by earlier contributors to this discussion. It can be used multiple times on the same culture or piece of tissue. It can be used to get an indication of apoptosis (in which case we follow up with apoptosis assays, such as TUNEL and caspases) and as a measure of cell proliferation. We have used it on many cell types, cell aggregates and tissue biopsies and found it to be very reliable provided that you do two things. 1) Check that you have a linear assay, we usually do this by varying the amount of biological material and 2) then hold the biological material constant and vary the amount of rezasurin. You should get the same answer at each concentration. When using the assay it is best to check the outcome at multiple time points in order to avoid coming to the wrong conclusion if your experimental procedure has pushed the cells down apoptotic pathways. In some cases we use DNA assays inorder to avoid overestimation of vibaility if the experimental treatment results in the detachment and loss of adherent cells.
Basically you need data from different assays...MTT, MTS, and Alamar blue assay is a good assay to start with. MTT and Alamar blue assays do not measure rate of cell proliferation or cell death-they only measure the cells which survive your drug or toxin. To measure rate of cell proliferation, one could use BrDU incorporation. FACS analysis of PI stained cells tells you how many cells are in different stages of cell cycle-you can find the mitotic fraction. For cell death, it is easy to count dead cells by Trypan blue exclusion assay or Annexin V staining. TUNEL assays or DNA ladder can also be used to measure apoptosis. There are assays for necrosis and autophagy-so its a project by itself! Hope this helps !
Great comments, I agree that care should be taken with alamarBlue and that at least one other assay should be used in parallel as described by Edita and Panagiotis. I usually use alamarBlue to compare the impact of experimental treatments. The first reading is used to determine viability and later time points relative proliferation. In the case of treatments on adherent cells combination with DNA assays is also important for viability assessment as well as proliferation because some cells may detach from the substrate due to the treatment and are not necessarily dead. In the case of tissues , like blood vessel rings, heart valve leaflets or biopsy samples, we would usually correlate the alamarBlue results with a functional assay or cell recovery protocol to make sure that the alamarBlue is giving valid results.
I dont have much of an input here, other than I was at a recent Promega event in which they highlighted MTT assays are not as reliable or accurate as people think, strange for them to bad mouth their own products. But our lab group found this to be true before the event. MTT did not reflect the viable cell count and for this reason we are using a cell death assay instead, one that measures dead cell protease. This is much more accurate, if you can use this assay type.
Hi Kirsty: Do you have a reference or protocol for a "dead cell protease assay". It sounds very interesting.
we use presto blue for stem Cell(hDPSC) viability and proliferation and results showed prestoblue much more easy,simple and affordable than mtt .
I have tried a variety of assays in an attempt to find a consistent and not overly time consuming method to get consistent data. What I have found with Alamar Blue, is while it is cheap and extremely easy to use (owing to it's low toxicity), it does need to be optimised specific to your cell line. If you cells are suspension lines, this makes it additionally a bit more complicated to optimise the time line.
I have a preference for MTT/MTS assay. They are not particularly time consuming and can be run in 96-well plates and in larger or smaller volumes dependant on sample size and repeatability options. This can often be coupled with an LDH assay to give you a secondary set of tests for confirmation.
To overcome some of the challenges with inconsistencies as well as in terms of having one or two highly metabolic cells in a well over proliferating cells, I normalise my data using a bicinchoninic acid assay (BCA) to measure protein content within the cells being tested.
Like everyone has stated already, it is a very good rule to use two or three different assay methods for cytotoxicity.
Good luck!
MTT based assays are a good first step. These assays are most widely used and give reliable estimates of viability for adherent cells. Can Kristy explain why Promega faulted this assay? I know MTT does not work well when cells are at low density. Redox agents in the media (like ascorbic acid) give false + in MTT. I would like to hear other criticisms on MTT assays . Hope this helps
For short time exposures, like 4 hours, whcih cell cytotoxicity assay is best, LDH or MTT?
I have used Alamar blue and it worked out well when cells were at low density. I'm going to use MTS and I have heard that it is a good assay and easy to use unlike MTT.
Presto blue is quite similar to Alamar blue, both of them are cell viability assays targeting the metabolic activity. But Presto blue is cheaper and easier to preform. Anyway It is good to indicate different assays that target different activities, for instance, Alamar blue and MTT or Alamar blue and MTS.
I received many inquiries about home-made Alamar Blue solution. Please read the protocol in my website.
http://www.cellsignet.com/protocols/buffers.html
"...great care must be taken in establishing the proper controls and parameters when using the MTT assay to count cells treated with drugs that alter respiration and metabolism. Cells exposed to uncouplers, such as 2,4-dinitrophenol, displayed a very large “false high” in cell number as compared to the same number of cells from the vehicle-treated control group (see Note 5)". Optimization of the Tetrazolium Dye (MTT) Colorimetric Assay for Cellular Growth and Viability by Paul W. Sylvester
See: http://www.ncbi.nlm.nih.gov/books/NBK144065/
for a guide.
Someone said that WST-1 is primarily reduced at the surface of cells (extracellular), while the reduction of MTT is taken place in the cells. Is it true?
@Edita Aksamitiene
Thank you for your home-made AB protocol. That's really money saving and I am going to try it. But I still have some Qs about the details. How long do you incubate the cells after adding AB? Will incubation of cells in PBS decrease their activity? Will be better if prepare 10X AB solution and add it directly into medium? Thank you!
I am currently having problems with the Quant-iT PicoGreen test for proliferation, does anyone have any thoughts on the reliability of this test? I am considering switching to MTS which I think might be a more robust alternative. Thank you!
Hi,
I have got different results from the same exp in between MTT and WST1 assay. According to the MTT results my compound is toxic but for the WST1 results it is not! I visualized the cells with microscope and saw some of them were dead. So do I need to try with another assay? Many thanks.
Dear Dr Tala,
In our studies toxicity evaluations were performed using two cytotoxicity assays instead of just one. This is because certain chemicals have been reported to give divergent results in different toxicity tests including the NRU and MTT assays (Olivier et al 1995; Chiba et al 1998). Besides, Evans et al (2001) have recently found that in some cases one of the NRU or MTT assays can be more sensitive in detecting the toxicity of non-viral transfection reagents.
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Evans, A.R., Alexander, D., Burke, P. and Reed, C.J. (2001) Toxicity and transfection efficiency: comparison between four commercially available non-viral transfection reagents in a human bronchial epithelial cell line. British Pharmaceutical Conference Science Proceedings 2001, 106.
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Evans, A. (2003) In Vitro and Ex Vivo Studies on the Toxicity and Efficacy of a Selection of Non-Viral Transfection Reagents. PhD Thesis, Liverpool John Moores University, Liverpool, UK.
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Please also see attached paper.
Zelal Adiguzel The same thing happened to me. For same compound, MTT shows it to be highly toxic whereas WST1 shows almost 100% viability. Same results remain after several rounds of repetition. I am wondering if you have figured out why such thing happened. Did you end up trying other assays? Thanks!
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