If you want to get any information about LD50 you need to perform an assay that counts dead cells: e.g. tunel assay or flow cytometry.

MTT assay, alamar blue assay, calcein assay, ... are all viability tests and do not give information about cell death but about viability.

MTT has the disadvantage that you can not check the viability at different time points since you have to sacrifice the cells where as alamar blue gives you the option to measure the viability of the same cell culture at different points of time.

You might like this book since it provides short descriptions, advantages and disadvanteges of the test being discussed in this forum:

http://books.google.de/books?id=nHmCmG7koSkC&printsec=frontcover&hl=de&source=gbs_ge_summary_r&cad=0#v=onepage&q&f=false

I would like to suggest you MTT assay for anti cancer drug because it is more convenient than trypan blue staining. Although both are used to check the viability of cells, MTT is colorometric assay which provides you the exact percentage of viability. It is hectic to count the viability through trypan dye and makes us suspicious regarding to the data. Also, in MTT assay you can use many drugs together which save your time.

Dear, generally MTT is the preferable method to determine IC50 for most of cancerous and noncancerous cells, and more reliable. While Trypan blue usually used for primary cell cultures that can not survive for long period outside the body and slow growing such as peripheral blood mononuclear cells. TQ

Try alamar blue. It is very sensitive and can be used colorimetrically or as a fluorescent assay.

The MTT assay is generally accepted for cell viability. To validate your MTT assay, you could count cells in response to the same conditions you used in your MTT assay.

Go for trypan blue...Its more reliable and reproducible than MTT assay.

If you want to get any information about LD50 you need to perform an assay that counts dead cells: e.g. tunel assay or flow cytometry.

MTT assay, alamar blue assay, calcein assay, ... are all viability tests and do not give information about cell death but about viability.

MTT has the disadvantage that you can not check the viability at different time points since you have to sacrifice the cells where as alamar blue gives you the option to measure the viability of the same cell culture at different points of time.

You might like this book since it provides short descriptions, advantages and disadvanteges of the test being discussed in this forum:

http://books.google.de/books?id=nHmCmG7koSkC&printsec=frontcover&hl=de&source=gbs_ge_summary_r&cad=0#v=onepage&q&f=false

I would also recommend alamar blue instead of MTT. MTT kills your cells and alamar blue will not, allowing you to do further analysis with the same cells.

The best assay is trypan blue. MTT and alamar blue are useful but their major drawback is their inability to distinguish cell toxicity (cell death) from cytostatic. affects. Trypan blue gives you total viable cells and % viability, thus allowing you to differentiate between toxicity and cytostatic

MTT is convenient and you can simultaneously check for multiple drugs, saving you time. alternatively you can use cell counting kit 8 (CCK8) for viability which is quick and accurate in my opinion.

if flow cytometry is available you can check the proliferation index by

Proliferation index (%) = (S +G2/M)/(G0/G1 + S + G2/M)X100%.

and,S and G2/Mphase cells for apoptotic cell death.

I think most people use MTT to determine IC50 but you have to bear in mind that is based on metabolization of the compounds. So, you can have false positives, because your cells can stop proliferation and metabolism but will be still alive and not death. If you can to be sure, combine the results with trypan blue.

Hi............I feel MTT is the best method,even we did MTT assay it is a simple method to chick cell viability.

hai... i think alamar is better than MTT because of sensitivity. but to know which concentration of drug effect in cancer cell MTT is better. i flow MTT specially for adherent cancer lines.

Dear Eric Hill

I agree with Daniel Medina, The best assay is trypan blue (than MTT and alamar blue ).

I would also like to add to those who are directing you to Trypan Blue. It's a relatively simple assay to do.

If you'd like a bit more information about the cell status, we have, in the past, used a propidium iodide/Annexin V co-stain to identify cells which were in early and late apoptosis. I guess whether this is of any relevance depends on the time course and the method you're using to look at cell death. Although time varies, apoptosis can take around 12-24 hours to complete (although you will see some evidence after a few hours). If you're looking quite early on, this is where the Annexin V assay will come in useful. You can buy it commercially, it's not too expensive.

I agree with Dean Kavanagh. I used a propidium iodide/Annexin V co-stain to dentify cells which died by apoptosis pathways. I described and compared three methods of melanoma cell viability estimation (trypan blue staining, MTT and propidium iodide/Annexin V co-stain) in my paper.

ApoTox-Glo™ Triplex Assay (viable, died cells, apoptosis)

MultiTox-Fluor Multiplex Cytotoxicity Assy (viable, died cells)

You might also like to look at these tests from Promega. These tests are independent of metabolism. Viable and dead cells are measured with the help of proteases who are available in dead and viale cells. For apoptosis the first assay measures the caspase 3/7 activities I think.

The assays are easy to perform with a fluorometer but are not cheap. If you want to buy them ask for discount. You usually get a high discount if you ask. Good luck for your experiments!

I think trypan blue is good because of its sensitivity and simplicity nature. If 'countess' automated cell counter is available it makes it better because of improved accuracy. It is what we use in our lab and is fine for determination of percentage cell death and viability, it takes only few seconds to 2 minutes to get your results . But literature said you could also use Sulforhodamine B colorimetric assay method to determine LD50 (Vichai and Kirtikara, 2006) .We use it for IC50 determination and plan to test it for LD50 determination in future .

To obtain a good measure of cell cancer proliferation or inhibition, Tripan blue method is a rapid and effective way.

MTT test give you a good estimation of the cell viability (not dead or alive cell count) but it is not easy to use it with cell suspensions (it is usually used for adherent cell cultures).

I'll go for CCK-8, It's very easy to use, sensitive and straight to the point. With it you can determine the viability of your cells at varied concentration within a limited period of time. However, the Kit is expensive. In the alternative you can use MTT Assay which will take an extra hour before you acquire your results. All the best!

Is the Guava viacount assay ( flow cytometry) will give the same result as MTT assay ? if I am looking for anti cancer drug ?

It's not about the MTT or cell viability, both are required for the different function. MTT assay determines the metabolism and proliferation process, whereas trypan determine whether  cell are dead or alive.

SRB (sulforhodamine assay) is a more advanced version of MTT assay for assessing in-vitro cytotoxicity which can give you better result with more reliability.

You need to perform the assay for each concentration of cytotoxic agent in triplicates with repetition assay for reproducibility to evaluate accurate IC50 value.

Also while withdrawing the media and replacing with DMSO to dissolve the dye in wells, you need to be utmost careful so that the dye will not come out and will dissolve completely in DMSO before colorimetric readout.

In my view, it does not matter whether you assess viability or cell death as when you assess one of the parameter, you will be getting the other one by default.

Trypan blue assay may not be a reliable (erratic results with assay repetition) assay and you cannot compare this assay with MTT or SRB assays that are more reliable in view of cytotoxicity assessment for Anti-cancer Drug Discovery Research.

Hope this helps.....

Best Regards,

Grandhi V Ramalingayya