I am conducting MTT assay of HCT-15 cells using a drug; concentration is 10uM. I am getting 75-80% cell viability. Actually i want to induce apoptosis in these by using this drug, but my professor is suggesting that this drug has some toxic effect on cells. He says that you should get cell viabilty upto 95% which will indicate that this drug doesn't have toxic effect on drug, rather it is inducing apoptosis. I will further do western blot for checking some apoptotic pathways. Please help me here that if i get cell viability 95% in MTT assay, will my drug have some down/up regulatory effect on proteins of apoptotic pathways? And tell me the range of MTT assay result regrding apoptosis. I hope you can pick my point.
Hi Salman.
First of all MTT assay doesn't show apoptosis, but rather decrease of cells viability or metabolic activity. Moreover if you think about toxicity maybe you should make additional controls? I usually make at least 3 samples (with repeats): control cells (only with growing medium), cells with the solvent of the drug like DMSO etc (with the same concentration (v/v) as in the sample with drug) and sample with drug, then you can check if the drug is actually effective.
For me 95% of cells viability in probe with drug is uncertain.. If you calculate average and SD it's quite probable that you wouldn't be sure if your drug really acts.
Next, you can imagine that if you have for example 1 mln of the cells in a very good condition, it is possible to obtain the same result in MTT assay as in situation of having 1.5 mln of the cells in worse condition. So you have to know also what is the growth rate of your material.
I agree with the previous opinions, western blotting (like PARP or caspase 3 cleavage), DNA fragmentation assay and FACS is better to know what you actually observe. I usually make MTT assay (because it's fast and cheep) at the begining to check if something is going on and if the results are interesting, then I make additional tests like FACS.
And one more, there is a mistake in Saeed heidari keshel answer: double positive cells annexinV/PI it's not a necrosis rather late apoptosis.
Ann+/PI- early apoptosis
Ann+/PI+ late apoptosis
Ann-/PI- live cells
Ann-/PI+ necrosis
Good luck! :)
You need to validate your MTT assay with an assay that measures for cell death, because if your cell line proliferates rapidly, the MTT assay will not be a clear indicator of apoptosis. I recommend performing an Annexin V - PI live cell death assay, as this will measure and indicate both apoptotic cells and necrotic cells.
1. Live cells - Annexin V negative, PI negative
2. Early death - Annexin V positive, PI negative
3. Late death - Annexin V positive, PI positive
4. Necrotic death - Annexin V negative, PI positive
Additionally, you can correlate this information for your drug with expression of pro or anti apoptotic gene/protein expression, but remember that increased levels do not automatically indicate death or survival. Other things to measure would be cleaved caspase 3 or PARP cleavage.
Hi, Salman, first, I would suggest you to determine specific drug concentration, ea. concentration of your drug which specifically interact with receptor. In this case you may not have a toxic effect. second, instead MTT assay you have to use viability determination with fluorescent dye, as Anne recommended.
Finally, you can try in situ TUNNEL assay to distinguish necrosis and apoptosis.
BUT, first, you must be sure that effect of the drug on cell is specific.
If you mean necrotic death as toxicity of the drug, then MTT is not the method of choice. As far as I know, MTT will show reduced viability of cells independent of whether it is apoptotic or necrotic. So the 20% death u see could be apoptotic also. If you want to rule out non-apoptotic effect / toxicity of the drug, better use a membrane integrity based assay (eg. Trypan blue stain, PI stain etc). Even though its a little complicated for adherent cells (to quantify), the above PI/Annexin double stain method proposed by Anne is very good to differentiate apoptosis.
The MTT assay measures metabolic activity (mainly succinate dehydrogenase). Therefore, some of your cells can be quiescent rather than dead. For best results, you should use a clonogenic assay to distinguish dead from living cells that may be metabolically inactive.
Although it is used to assess cell viability, MTT assay measures the cellular metabolic activity. Therefore, you need a death assay for your purpose. To differentiate between necrosis and apoptosis, EM gives a definitive answer (along with studying the known death signaling pathways)
MTT assay is an evaluation of cell viability on certain treatment, but not a specific test for apoptosis. A high value could be that you had more necrosis directly induced by your stimulus or in secondary to apoptotic death. For screening apoptosis, according to the experience of some labs, at least three different methods should be employed in order to confirm the induction of apoptosis. A reliable test for cell morphology could be microscopic view of fragmented nuclei; flow cytometry-based measurement of phosphatidylserine turnover, and drop of mitochondrial membrane potential, and assay of cleavage of caspases and degradation of PARP using Western blotting, and of DNA laddering are also very useful.
MTT is a colorimetric assay for determining cell viability however for determining apoptosis, Annexin V-PI is a better assay as suggested by Anne. Also PI-RNase may be used. The expression of pro and anti apoptotic proteins like Bax, BCl2, BClXL etc may also be assessed by Western blot/FACS based studies to get a better idea.
MTT assay measure metabolic activity then it indicate you Cell viability. First, you need a control without drug to compare the toxicity of the drug. As other people comment you MTT assay is not specific for determining Cell death (apoptosis or necrosis). Annexin V- PI assay as suggested by Anne is good but Live cells are negative for both Annexin V and PI. However as suggested
MTT assay is not sufficient to fully assess cell viability, you have to use another assay such as LDH release and-or trypan blue exclusion. If you got mortality you should first test a dose response curve of your product or drug to check for toxicity and then try a time course. A very short response time would speak in favour of necrosis whereas delayed cell death will probably indicate apoptosis. This assumption would therefore be verified by more specific assay (annexin, tuneL...)
With the MTT assay is not possible determinate if your drug induce apoptosis or necrosis, only you know how much affect the cells viability. To discriminated this process, Is necessary an study of FACS with Annexina V-IP (at 4°C and 37 °C ). If the death decreased with the low temperature is indicative of a necrosis process. But if you desire confirmer of these results, a caspasas 3 assay is the better option.
Dear first thing is that MTT assay is just anti-proliferative assay. It does not indicate whether cells are undergoing apoptosis or necrosis. The other important point is, no drug till now is available as you think that can induce apoptosis while the cell viability remains 95%. If 95% cells are alive, where is the effect of apoptosis then.
please see this link:http://www.bioscience.co.uk/userfiles/pdf/Apoptosis_Necrosis_and_Cell_Viability_Assays.pdf
In the following, I suggest to used more simple assay like trypan blue, which measures rupture of the PM (necrosis and late necrosis after apoptosis) or more sophisticated ones, like the combination annexin V /propidium iodide with cytofluorimeter. Single positive cells for annexin V (apoptotic) double positive cells annexinV PI (necrotic).
Hi Salman.
First of all MTT assay doesn't show apoptosis, but rather decrease of cells viability or metabolic activity. Moreover if you think about toxicity maybe you should make additional controls? I usually make at least 3 samples (with repeats): control cells (only with growing medium), cells with the solvent of the drug like DMSO etc (with the same concentration (v/v) as in the sample with drug) and sample with drug, then you can check if the drug is actually effective.
For me 95% of cells viability in probe with drug is uncertain.. If you calculate average and SD it's quite probable that you wouldn't be sure if your drug really acts.
Next, you can imagine that if you have for example 1 mln of the cells in a very good condition, it is possible to obtain the same result in MTT assay as in situation of having 1.5 mln of the cells in worse condition. So you have to know also what is the growth rate of your material.
I agree with the previous opinions, western blotting (like PARP or caspase 3 cleavage), DNA fragmentation assay and FACS is better to know what you actually observe. I usually make MTT assay (because it's fast and cheep) at the begining to check if something is going on and if the results are interesting, then I make additional tests like FACS.
And one more, there is a mistake in Saeed heidari keshel answer: double positive cells annexinV/PI it's not a necrosis rather late apoptosis.
Ann+/PI- early apoptosis
Ann+/PI+ late apoptosis
Ann-/PI- live cells
Ann-/PI+ necrosis
Good luck! :)
HI Salman, MTT assay measures metabolic activity of the cells. This assay is very preliminary. Low viability in MTT assay might be due to anti-proliferative or apoptotic or necrotic effect of your compound. To rule out these effects, you need to do more experiments related to them. For anti-proliferative effect, you can do cell cycle analysis using flow or expression of cell cycle regulators using western. For apoptosis and necrosis, you can stain both untreated and treated cells with annexin and propidium iodide. Moreover, I suggest you to check the morphology of the cells regularly after treatment with that particular molecule. Always use the proper control like the solvent where you dissolved your molecule.
All the best !
Raj
Hi Salman!
I strongly believe with Emilia Joanna. I am always carrying out my tests as Emilia is doing and said:
"Usually make at least 3 samples (with repeats): control cells (only with growing medium), cells with the solvent of the drug like DMSO, ethanol, Nanoparticles, etc (with the same concentration (v/v) as exist in the sample with drug) and sample with drug, then you can check if the drug is actually effective".
When you have 75-80% of viability, it is indicated that something in your supplement is toxic even having low toxicity. Maybe it is your solvents or your drug only. So please check the controls with or without solvent as Emilia recommended.
By MTT test you will not be able to examine the apoptosis, late apoptosis or necrosis. MTT is just gives you the result for viability and the rate of cell death not kind of it. For this purpose you can also examine LDH or ROS of the cell.
As other contributors’ recommended, I am also suggesting you to use Annexin V/PI either with flowcytometry or fluorescent microscopy. You can also use Rhodamine which is a green fluorescent dye for apoptosis test.
The description of apoptosis, late apoptosis/necrosis which Emilia said is truthful.
“Ann+/PI- early apoptosis
Ann+/PI+ late apoptosis
Ann-/PI- live cells
Ann-/PI+ necrosis”
And for the final analysis, reaped your work in triplicate and calculate SD and Error Barr, so your experiments would be significant.
Maybe this file is useful for you.
http://www.bioscience.co.uk/userfiles/pdf/Apoptosis_Necrosis_and_Cell_Viability_Assays.pdf.
Hope you can cope with your test and drug.
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