Cannot you google the answer? There must be detailed description of the method online.
Of course it is possible to google the aswers, and after several months you will be able to do MTT perfectly with kit-8 or without. Only practice shows there are many people who suceeded in googling but haven't succeeded in MTT unfortunatelly...
Dear Nabil,
MTT is universal for all cell cultures viability-cell death test, and it is rather simple because you practically need 4 things for it: 96-plates, steril solution of MTT reagent and DMSO for fixation-counting in plate reader. AND also cell culture which you grow properly that it doesn't die by itself instead of doing so because of treatment...
Kit-8 is the same MTT, only it is more expensive to buy it in box instead of weighting-dissolving tetrazolium bromid (MTT) reagent yourself.
1. First thing you do is to learn how to passage cells into 96-well plate. The reason here is your curves are as much presentable as more difference you demonstrate between your "+" and "-". So, you better place your cells for incubation with treatment for 72-96 hours than for 24. Therefore you should evaluate your current culture proliferative activity to make out how many cells suspension concentration feels comfortably during incubation of 4-5 days in wells without changing growth medium. In average it is about 1-1.5 mili of cells for one 96-plate in 20ml growth medium. Or approximately 2/3 or the whole 25mm flask MCF7 as they are Insuline + and proliferate slowly, 1/2 or 2/3 of flask Hep-G2 as they multiply more vividly.
You poor 200 mkl of your culture suspension (from one steril bottle!) sterilly into every well; for 96-plate it is possible to use 8-channel pipette, but I make it will single channel to be more precise. Believe, after many hundred of tests it is possible to make 96-well within 7 minutes. Incubation before anti-tumor agent and MTT reagent application is normally 24 hours, but it also depends on cell culture proliferative potential - your cells should be in log phase of cycle in treament start for maximum effect.
I prefere flat-bottom 96-plates, but for suspension cultures it is possible to use dom-bottom ones also.
2. You prepare MTT reagent (or take ready-made from kit-8 bottle). As far as I remember it is 5mg/ml for stock, so you weight this stuff in glass for 10-50 ml of solution and add water. I will check concentration tomorrow in dairy and type it to you. Few drops of PBS in the beginning will make yellow stuff to dissolve more quickly. Then you take 0.2 mkm filter and syringe and poor the solution into steril brown glass or 15ml falcon tubes. The solution is light-sensitive, wrap it into aluminium film if steril brown glass bottles are not available.
MTT stock is ready to drop 7-10 mkl into every well.
3. You should add treatment immediatelly after or just before MTT. For example, for anti-tumor agents it is optimal to make gradient of 8-12 concentrations with control of untreated cells. And expectable IC50 should be in the middle of gradient, and in the end - 100 percent cell death concentration. 2-4 wells of the same concentration is necessary to do for statistical validation of results.
Make drawing of concentration gradient in your dairy before the experiment, it will be necessary for addition of your reagent as well as for result's analysis.
Stupid people think it is possible to measure only cell cycle variations or smt. like antracyclines dosage cell death gradient. I measured antibody-targeted delivery with 3 cytotoxicity parameters at once, and gene thearapeutic constructions efficiency, and phosphokinases inhibitors activity, and photodynamic polymer-applied agents activity with laser beam activation, with ultraviolet activation - everything is possible when you LEARN to HANDLE the method instead of googling it.
4. After 48-96 hours of incubation in CO2 you sip out growth medium and poor 100 mkl DMSO into plates, and go to reader. For suspension growing cultures it is better to use 10 min 1200-1500g plates centrifugation (if centrifuge with plates adaptors is available) and to remove medium before DMSO.
YOUR MTT IS DONE. Trichloroacetic acid is not required in any step, I don't know for what some people use it in MTT.
Will check and send you MTT stock concentrations.
Greetings,
E.F.
Dear Nabil,
MTT reagent stock concentration is 5 mg/ml. So, for one 96-well assay you will use about 2 ml of the solution. I prepared for 10 to 50 ml, easier to push through 0.2 mkm membrane filter. MTT stock can be stored at +4 C for several weeks and even months.
But I am sorry, I told you wrong info when to add it and for how long. In my 10 years ago protocols I added it 10 mkl/well of plate for 4 hours during the last day of incubation in CO2 incubator, 48-96 hours past cytotoxic cell's treatment. Understandable as it is light-sensitive substance. By the end of these 4 hours you should see that alive cells have got violet color of farmazan crystalls, and the higher is cell death rate, the less is this color presented.
Forgot, paper works for years makes people stupid...
Good luck and greetings,
E.F.
Dear Elena, your answer is amazing! Only one tiny thing i disagree with is the storing of MTT solution. I did it 2-3 times (one time at +4 and 2 times on -20) and i have to repeat my experiments again. For eg. in untreated wells the absorbance was much lower than in those wells where no cell's were seeded, not to mention the treatment wells....
Dear Nikola,
There are about 10-15 different tetrazolium salts, they might be different in storage. What I always used was bromide. Dissolving in distilled water is low enough, if crystals of yellow salt is swimming in solution, or solution is not transparent, you should make new portion and poor few drops of DMSO or 1-2 ml PBS and vortex it before adding water to full volume. Solution should be bright yellow color and transparent before 0.2 mkm filtration. Storage in darkness is important, use aluminium film to cover vessel. If crystals appear during storage, or solution is not limpid - this solution is not good anymore. Mine was stable for 1-2 months, maybe even longer, and absorbance did not change.
Happy new year!
Spectrophotometric MTT assay
3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (Sigma-Aldrich) was dissolved in RPMI 1640 medium, at a concentration of 5 mg/ ml and fi ltered through a 0.2 μm filter. Subsequently, 100 μl of the yellow MTT solution was added to each well of a well plate, containing 1 ml of the cell suspension. The cells were then incubated at 37°C with 5% CO2. A blank solution was prepared according to the above procedure using complete medium without cells. After a three-hour incubation period, the resulting formazan crystals were dissolved using 1 ml of acidified isopropanol (0.05 N HCL in absolute isopropanol), and the absorbance of the obtained solution was measured at a wavelength of 570 nm using a spectrophotometer .
If dissolved in RPMI one would need to make new MTT reagent every time for sure. Cytotocicity test is not being made in 1ml volume (48- or 24-well plates) because reader does not accept these format; to replace DMSO, isopropanol or any other solvent to cuvettes or 96-plate is additional unnecessary step.
Acidified alchohole (isopropanol) is not dissolving cell membranes completely, it is fixator. So, absorbance rate will be partially lost in formazan which would not leave cells, and another portion of absorbance color - in not removed medium. The same happens with trichloracetic acid usage I think.
I really appreciate the responses by Elena who took a signifcant piece of her time to provide this very nice response.
In this line of explanation, I would drawn the attention of everyone that "MTT colorimetric assay" does not measure cytotoxicity through its sole IC50/GI50 concentration.
Cytotoxicity means "cell killing effects".
A colorimetric assay can only bring "relative global growth inhibition information" because it is a relative test in which you compare the ODs of a treated cell population to the ODs of a control condition (untreated cells) arbitrarily scaled at 100%.
When you obtain a concentration (for a given compound) decreasing by 50% the global growth (after x days), i.e. the GI50 concentration (or the IC50 as commonly used in the literature) you do not know whether your compound of interest killed 50% of the cells (cytotoxic effects), whether it inhibited 50% of the cell proliferation (cytostatic effects), whether it detached 50% of the cells (anti-adhesive, i.e. "in vitro antimetastatic" effects), etc..., etc...
Once you have determined the GI50 / IC50 concentration for a given compound on a given cell line, you must use complementary biochemical and/or morphological techniques to determine whether your compound is cytotoxic, cytostatic, anti-adhesive, etc..., etc...
The two attached articles by Galluzzi and colleagues (2012, 2015; Appendix-1 and Appendix-2) are of great help in this domain.
The attached article by Kornienko et al. (2013; Appendix-3) reviewed various chemicals that are able to induce non-apoptotic cell deaths in cancer cells.
Coming back to the IC50 / GI50 values obtained by means of a colorimetric assay (as for example the MTT one):
in the Mathieu et al. (2009 (Appendix-4) and 2015 (Appendix-5)) articles, the MTT test-related GI50 concentrations relate to actual cytotoxic effects.
In the Lefranc – Nunzo et al. (2013; Appendix-6) article, the MTT test-related GI50 concentrations relate to cytotoxic effects that in turn do not relate to apoptosis … This means that each cytotoxic effect does not “universally” translate into pro-apoptotic ones.
In the Van Goietsenoven et al. (2010; Appendix-7) article, the MTT test-related GI50 concentrations relate to cytostatic effects, neither to cytotoxic nor to pro-apoptotic ones.
Be aware that you cannot always translate the MTT test-related growth inhibition of a given compound into a precise GI50 value. Some compounds reach a “plateau” of inhibition (see Lefranc – Nunzo et al., 2013; Appendix-6).
Lastly, you can also have "false" data generated with the MTT colorimetric assay (see the attached article by Chan et al. (2013; Appendix-8) and the first NCI-60-cell line-related article (Alley et al., 1988 (Appendix-9)).
The US NCI set up a fantastic tool to characterize the effects of a given drug in terms of growth inhibition in a panel of 60 cancer cell lines belonging to >10 histopathological types (Alley et al., 1988; Appendix-9).
The US NCI clearly defined by means of the combination of the GI50 (growth inhibition), the LD50 (lethal dose by 50%) and the TGI (total growth inhibition) how to make the difference between a cytotoxic and a cytostatic compound:
The US NCI-related GI50 value corresponds to a global growth decrease by 50% induced by a compound on a given cell line “x” days after having cultured the cells with the drug and in comparison to an untreated control condition (= 100%) grown during the same time;
The US NCI-related LD50 value corresponds to the a global growth decrease by 50% induced by a compound on a given cell line “x” days after having cultured the cells with the drug and in comparison to the initial number of cells in the untreated control condition;
The TGI is the US NCI-related parameter to determine the concentration needed to kill 100% of the treated cells.
It is by comparing the GI50 to the LD50 value that one can determine whether a compound is cytotoxic or cytostatic, and not at all with the sole GI50 value.
We are using morphological approaches in my research unit to determine whether a compound is cytotoxic or cytostatic (see Lefranc-Nunzo et al., 2013; Mathieu et al. 2009 (Appendix-4), 2015 (Appendix-5); Van Goeitsenoven et al., 2010 (Appendix-7)).
The US NCI is not so far for having tested about one million of anticancer drugs, whose data are publicly available on the NCI website https://dtp.cancer.gov/databases_tools/data_search.htm
I actually benefited several times from the amazing help of the NCI in identifying the mechanism of action of an innovative anticancer compound (see for example Frederick et al. JMC 2011 (Appendix-10)).
Hoping that this long explanation would not be too boring,
Best regards
Robert
Dear Nabil,
Cell counting kit- 8 could be used for any type of cell. You can use it to assess the proliferative activity or viability of the cells of interest. Invariably you do not need pre-incubation while using the Kit. It works pretty similar to the commonly use MTT assay, with the only difference of having the end product of the reaction been water soluble. As such there is no need of additional usage of other agents for dissolution of the end product (formazan in the commonly use MTT).
The cell number that seems appropriate should fall within the 0.75-1.25 optical density (OD) which for me happens to always fall within 1-1.5 millions cells per ml, but this I believed it will depend on the type of cells you are using.
Dear Robert,
I appreciate you so much. Your answer had been what i was suspecting. It can not give us the absolute meaning of IC50 but a relative meaning of cell viability. Now i am relieved to know that
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