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 JoVE Biology

Cellular Toxicity of Nanogenomedicine in MCF-7 Cell Line: MTT assay

1,2, 1,3, 1, 2, 1,3

1Research Center for Pharmaceutical Nanotechnology, Faculty of Pharmacy, Tabriz University (Medical Sciences), 2Gifted and Talented Students Office, Educational Development Center, Tabriz University (Medical Sciences), 3School of Advanced Biomedical Sciences, Tabriz University (Medical Sciences)

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    Summary

    The MTT assay is an easy and reproducible colorimetric assay for evaluation of cell viability based on reduction of yellow MTT and production of water insoluble purple formazan. Here, the viability of MCF-7 cells upon treatment of nanogenomedicine has been evaluated.

    Date Published: 4/03/2009, Issue 26; doi: 10.3791/1191

    Cite this Article

    Ahmadian, S., Barar, J., Saei, A. A., Fakhree, M. A. A., Omidi, Y. Cellular Toxicity of Nanogenomedicine in MCF-7 Cell Line: MTT assay. J. Vis. Exp. (26), e1191, doi:10.3791/1191 (2009).

    Abstract

    Cytotoxicity of the futuristic nanogenomedicine (e.g., short interfering RNA and antisense) may hamper its clinical development. Of these, the gene-based medicine and/or its carrier may elicit cellular toxicity. For assessment of such cytotoxicity, a common methodology is largely dependent upon utilization of the 3-(4, 5-Dimethyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium bromide (MTT) assay which has been widely used as a colorimetric approach based on the activity of mitochondrial dehydrogenase enzymes in cells. In this current investigation, MCF-7 cells were inoculated in 96-well plate and at 50% confluency they were treated with different nanopolyplexes and subjected to MTT assay after 24 hours. Water soluble yellow MTT is metabolized by the metabolically active cells to the water insoluble purple formazan, which is further dissolved in dimethylsulfoxide and Sornson s buffer pH 10.5. The resultant product can be quantified by spectrophotometry using a plate reader at 570 nm.

    Protocol

    MCF-7 seeding in 96-well plate:

    MCF-7 cells were cultured in 25 t-flask in medium containing Dulbecco’s Modified Eagle’s Medium (DMEM), 10% FBS, 100 U/ml penicillin, and 100 μg/ml streptomycin at 37°C with 5% CO2, 95% air and complete humidity. Once reached ~90% confluency, they were detached using 0.05% trypsin/EDTA and counted by means of trypan blue and hemocytometer. These cells were then resuspended at a concentration of 4×104 cells/cm2 and added onto 96-well plate (i.e., 250 μl/well) by an 8-channel pipette For background absorption, some wells were remained cell-free, i.e. as blank control.

    Treating cells with different nanopolyplexes:

    At 40-50% confluency (48 hours post seeding), the cultivated cells were treated with nanostructured starburst polyamidoamine dendrimers (i.e., Superfect® and Polyfect®) and a novel test polymer following the transfection instruction provided by supplier. Cells were also treated with EGFR and scrambled antisense alone, and with the three different nanopolyplexes of these two oligonucleotides and with polymers (n=4). Four wells were remained untreated as control. After 4 hours the treatment media were removed and replenished with fresh media.

    MTT assay for evaluating cell viability:

    MTT assay was performed 24 hours after transfection. For this purpose, MTT solution was prepared at 1mg/ml in PBS and was filtered through a 0.2 µm filter. Then, 50 µl of MTT plus 200 µl of DMEM without phenol red were added into each well, except the cell-free blank wells. Cells were incubated for 4 hours at 37°C with 5% CO2, 95% air and complete humidity. After 4 hours, the MTT solution was removed and replaced with 200 µl of DMSO and 25µl Sorenson’s glycine buffer (glycine 0.1M, NaCl 0.1M, pH:10.5 with 0.1 NaOH). The plate was further incubated for 5 min at room temperature, and the optical density (OD) of the wells was determined using a plate reader at a test wavelength of 570 nm and a reference wavelength of 630 nm.

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    Discussion

    The MTT assay is deemed to be a versatile method and accordingly the viability of the cells could be evaluated upon various treatments. The production of resultant formazan appears to be proportional to the level of energy metabolism in the cells. Therefore, it is possible to measure the metabolically activated cells even in the absence of cell proliferation. The amount of formazan produced is proportional to the amount of MTT in the incubation medium. While, the concentration of MTT which is required to achieve maximum amount of formazan produced may change upon utilization of different cell lines Besides, having used this assay, very small number of living cells could be detected and the incidence of errors would be minimal since there is no need for washing steps. The absorption of formazan varies with cell number as well as pH which could be overcome with addition of buffer at pH 10.5 1. The color of formazan is stable for a few hours at room temperature 2. In the case of more than one plate, controls should be included in other plates as well. Nevertheless, this method suffers from some minor disadvantages: a) metabolically inactive cells cannot be discriminated with dead cells 3, b) MTT solution should be protected from light even though it could be stored at 4°C for a maximum of one month 2, c) it fails to validate drug stability in the medium, and d) cells used for MTT can not be subsequently used for any other assays.

    It should be evoked that phenol red absorbs at 570 nm. Further, it has been previously reported that the phenol red possesses estrogen activity which may affect the cell growth pattern within some estrogen responsive cells, ensuing imprecise MTT results. To avoid such impact, we have utilized DMEM without phenol red 4.

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    Disclosures

    Acknowledgements

    The authors would like to acknowledge Stem Cell Technology and Shahid Ghazi Tabriz companies respectively for providing DMEM without phenol red and sterile water for injection as gifts.

    Materials

    Name Company Catalog Number Comments
    25 t-flask Orange Scientific 5510100
    PBS Sigma-Aldrich P3813
    Dulbecco’s Modified Eagle’s Medium - high glucose Sigma-Aldrich D5671
    MTT Sigma-Aldrich M2128
    FBS Sigma-Aldrich F2442
    Penicillin 200,000 u/ml CinnaGen CR7913
    Streptomycin 200 mg/ml CinnaGen CR7912
    trypsin/EDTA Sigma-Aldrich T3924
    trypan blue Sigma-Aldrich T8154
    Superfect Qiagen 301105
    Polyfect Qiagen 301305
    EGFR antisense Eurofins MWG Operon
    Scrambled antisense Eurofins MWG Operon
    DMEM without phenol red GIBCO, by Life Technologies 11880
    Glycine Sigma-Aldrich G8898
    hemocytometer HBG
    8-channel pipette TreffLab Transferpette 96.9918.10.1
    Disposable syringe filter Iwaki, Asahi Techno Glass Corp. 2052-025
    Plate reader equipped with 570 nm &630 nm filters BioTek ELX808
    96-well plate with lid (flat bottom) Iwaki, Asahi Techno Glass Corp. 3860-096
    Laminar flow hood Esco Products
    CO2 Incubator ASTEC

    References

    1. J. A. Plumb, R Milroy, and S. B Kaye, "Effects of the pH Dependence of 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium Bromide-Formazan Absorption on Chemosensitivity Determined by a Novel Tetrazolium-based Assay," Cancer Research 49, 4435 (1989). Ref Type: Journal
    2. T. Mosmann, "Rapid Colorimetric Assay for Cellular Growth and Survival: Application to Proliferation and Cytotoxicity Assays," Journal of Lmmunological Methods 65, 55 (1983). Ref Type: Journal
    3. M Yang, et al., "Energy and Redox States in the C6 Glioma Cells Following Acute Exposure to Zn, Se +4 , and Se +6 and the Correlation with Apoptosis," Toxicology Mechanisms and Methods 16(1), 13 (2006). Ref Type: Journal
    4. D. M. Spinner, "MTT Growth Assays in Ovarian Cancer,"in Ovarian Cancer: Methods and Protocols, edited by Bartlett and .M.S. (Humana Press, Inc., 2000), pp.175-177.

    Comments

    22 Comments

    what happen if MTT more than 4 hrs. 
    Reply

    Posted by: AnonymousApril 4, 2009, 12:40 PM

    time of incubation is one of influencing factors and could vary from 30 min up to 48 hours. it could be optimized according to the cell line
    Reply

    Posted by: AnonymousApril 7, 2009, 9:56 AM

    I am writing to appreciate about your useful video!
    Reply

    Posted by: AnonymousApril 8, 2009, 5:21 AM

    thanks! it is useful
    Reply

    Posted by: AnonymousApril 8, 2009, 5:26 AM

    very useful and practical, thank you.
    Reply

    Posted by: AnonymousMay 26, 2009, 2:46 PM

    Thank you for your very useful video :)
    Reply

    Posted by: Taksanee M.June 16, 2009, 11:47 PM

    why you do not open the lid of 96-well plate to detect OD value?
    Reply

    Posted by: AnonymousJune 24, 2009, 4:54 AM

    very useful, thanks alot
    Reply

    Posted by: AnonymousJuly 8, 2009, 5:24 PM

    what is the significanse of the MTT ASSAY? (in brief)
    Reply

    Posted by: AnonymousJuly 18, 2009, 4:39 AM

    Can you determine the cell proliferation with MTT assay?
    Reply

    Posted by: AnonymousApril 14, 2011, 10:52 PM

    سلام
    ž²;سته نباشید ž²;انم احمدیان.میتونید از نرم افزار آی دی ام استفاده کنید
    به امید موف ²;یت iran
    Reply

    Posted by: mohsen j.May 13, 2012, 9:37 AM

    سلام ž²;انم احمدیان
    و ²;تتون بž²;یر.من دانشجوی کارشناسی ارشد سم شناسی دانشگاه علوم ¦²;زشکی مازندران هستم.موضوع تز من ارزیابی سیتوتوکسیسیته یک نانو دارو میباشد.ž²;واستم از محضرتان ž²;واهش کنم که لطفا ویدئو ام تی تی رو برام بفرستید چون هر کاری میکنم دانلود نمیشه.با س¦²;اس و احترام فراوان از شما مح ²; ²; ž²;وب و برجسته
    Reply

    Posted by: AnonymousMay 24, 2011, 2:19 AM

    thank you so much for the video, really useful and clearified a lot of things for us!
    Reply

    Posted by: AnonymousMay 26, 2011, 3:21 AM

    Hi
    thank you very much dear dr.omidi for your attention,mer30
    Reply

    Posted by: AnonymousMay 27, 2011, 11:35 PM

    سلام و ²;تتون بž²;یر. مطالب ž²;یلی جامع وجالب بود. لطف می کنین ویدو ام.تی.تی رو برام بفرستین
    ¦²;یشا¦²;یش از مساعدتتون کمال تشکر رو دارم.
    Reply

    Posted by: AnonymousJuly 7, 2011, 4:11 AM

    Hi, Is it really necessary to treat the cells with the compound of interest(chemicals we are testing) for ²4 hours.
    Cant we just treat them for the required time period.For eg, if in my experiment the compound of interest will be in contact with cells for 6 hours then i think MTT assay should be done for 6 hours.Am i right.Any help will be greatly appreciated
    Reply

    Posted by: AnonymousOctober 3, 2011, 8:44 AM

    Timing issue is based upon your work. You can do MTT after 6, 1², ²4 hr depend on your experiment goal and effectiveness of materials used.
    Reply

    Posted by: AnonymousOctober 3, 2011, 9:51 AM

    Hi, i did MTT assay as per the protocol and my optical density readings in control was 0.1.Do you think its right.Please help
    Reply

    Posted by: AnonymousFebruary 16, 2012, 9:48 AM

    Thank you very much Dr.Omidi.I really appreciate it
    Reply

    Posted by: AnonymousOctober 5, 2011, 4:33 AM

    If the cells are grown in DMEM F-1² media containing phenol red ,would this affect the results? Also what formula would you suggest for calculating percentage cell growth after treatment with a compound
    Reply

    Posted by: AnonymousOctober 18, 2011, 7:06 PM

    why did you use Sorenson²17;s glycine buffer when solving the dyeA²9²;and is that better than using DMSO alone?
    Reply

    Posted by: AnonymousMarch 19, 2012, 5:05 AM

    please send this video for teaching purpose
    Reply

    Posted by: SARVESH S.December 24, 2013, 9:04 PM

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