Product Information Sheet MarkerGene Non-Radioactive Cell Proliferation Assay Kit (MTT Assay) Product M1475 Marker Gene Technologies, Inc. University of Oregon Riverfront Research Park 1850 Millrace Drive Eugene, Oregon 97403 1-888-218-4062 www.markergene.com
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Marker Gene Non-Radioactive Cell Proliferation Assay Kit (Product 1475) NOTE: The following information is given as a viable methodology for use of the Marker Gene Non-Radioactive Cell Proliferation Assay Kit. The user may determine their own best methods for use dependent on the specific conditions present in their experiment. I. OVERVIEW Reduction of tetrazolium dyes by both prokaryote and eukaryote cells is commonly used to test for cell viability and to estimate cell growth. The MarkerGene Non-Radioactive Cell Proliferation Assay Kit uses a common tetrazolium dye MTT ((3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) as a method of quickly and accurately measuring cell viability. The assay relies on the reduction by live cells of the tetrazolium salt MTT by intracellular oxidoreductases. It is therefore widely regarded as an accurate indicator of cell redox activity. The MTT dye exhibits a pale yellow color, but is reduced intracellularly to a deep purple formazan product (see Figure 1). It is widely accepted that the reduction occurs in the mitochondria, although evidence exists that reduction of MTT can also occur in other areas of the cell. 9 Figure. 1: MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) is reduced in viable cells by action of Mitochondrial Reductase to a purple formazan. 3
In order to monitor cell growth, cytotoxicity, or other cell parameters, cells are grown in the presence of test compounds, drugs or other factors for a period of between several hours to several days. The media is removed and the cells washed with an isotonic buffer (PBS) followed by addition of a MTT staining solution. Since the formazan product precipitates in the cells, a DMSO based solubilization solution is used to dissolve the insoluble formazan to yield a purple colored solution. The absorbance of this colored solution is then quantified using a plate reader or spectrophotometer with absorbance at 570nm. The protocol gives specific details on running the assay, along with methods for quantitating the number of cells/well, blanks for non-treated cellular samples, as well as representative data. For further information regarding the assay protocol or methods, please visit our website at www.markergene.com, telephone our technical staff at 1-888-218-4062 or email us at techservice@markergene.com. The absorbance spectrum of formazan in Solubilization Solution. 4
II. MATERIALS This kit contains enough reagents to perform: - 20 full-plate assays in a 12-well format - 20 full-plate assays in a 6-well format - 12 full-plate assays in a 96-well format - 48 60mm plate assays - 24 100mm plate assays A.) B.) MTT (M1475-001): Unstable in solution. Prepare MTT Reaction Buffer solution as needed before each use. MTT Buffer(PBS) (M1475-002): Buffer solution for making MTT Reaction Buffer. C.) HEPES (M1475-003): ph Buffer for addition to working media. D.) Solubilization Solution (M1475-004): Sorenson s Glycine Buffer in DMSO. For dissolving formazan to give colored solution. E.) Aluminum Foil or equivalent (Not Provided): For wrapping plates to light-tight them during the incubation period. F.) Growth Media (Not Provided): Tissue culture media for making buffered working media with addition of HEPES. Storage and Handling. Refer to kit contents table for storage conditions. Some materials are light sensitive and may need to be stored chilled. All materials should be handled with care. Unopened reagents should be stable for at least 6 months following purchase. Please consult our technical assistance desk for more information about use and storage conditions. 5
III. ASSAY CONDITIONS Recommended Plates: - Falcon 353043 12-well Tissue Culture plate - Falcon 353077 Microtest 96-well Tissue Culture plate - BD Falcon 100 x 15 mm Tissue Culture Petri Dish - Corning 60mm or 35 mm Tissue Culture Dish or similar. All transfer of cells between plates, and addition and removal of reagents should be done under a laminar flow hood. Plates will potentially be incubated after transfer and leading up to the actual assay, and as part of the assay itself after addition of reagents. It is recommended that measurements be made in triplicate, if possible. The assay monitors absorbance at 570nm using a microplate assay format. Other filter sets with similar wavelengths can be used. The user is asked to consult with the manufacturer (or instrument manual) for the particular instrument in use for appropriate setup conditions needed for these measurements. NOTE: The assay is carried out on cells cultured in a microtiterplate. When plating cells out into the plate wells, initial cell counts per well should be adjusted according to the amount of expected incubation time before the assay is carried out. A starting point for ideal cell numbers is a final concentration between 5 15 x 10 3 cells/ml in each well for respective incubation times between 24-72 hrs from the time cells are counted into the plates. (Determined using NIH3T3 incubated at 5% CO 2 ). 6
IV. GENERATION OF A STANDARD CURVE: In order to translate absorbance values into viable cells / well values, a standard curve relating absorbance to cell number needs to be generated as follows: 1.) Count cells into wells of microtiter plate. a. Aspirate off media, wash cells with PBS or appropriate isotonic salt buffer, trypsinize, and resuspend in 1 ml of complete growth medium. b. Count cell suspension using a hemacytometer. Make sure that the suspension is homogenized. It is recommended that multiple counts are taken to ensure statistical accuracy. c. Dilute volumes of cell suspension directly into wells of plate in complete growth medium to achieve an appropriate gradient of concentrations. Do this in triplicate. Dilute the first set 1:10, the second 1:100, the third 1:1000, etc. (See Table 1). d. For a shallower curve dilute 1:2, 1:4, 1:8, etc. e. The standard curve should be designed such that the cell number on which the proliferation assay will be carried out at exists within the data range of the curve. For example in the below table 1 the standard curve produced by this distribution would be suited for use with proliferation assays run with cell counts between 6x10 1 and 6x10 3 cells/ml. f. Include a zero cell blank set for the zero point background adjustment. Subtract the value produced by this set of wells to give corrected absorbance. 7
Table 1: 12-Well plate of triplicate sets. Exemplary Data. 6.0 x 10 3 6.0 x 10 2 6.0 x 10 1 No (Background Control) 6.0 x 10 3 6.0 x 10 2 6.0 x 10 1 No (Background Control) 6.0 x 10 3 6.0 x 10 2 6.0 x 10 1 No (Background Control) 2.) Allow the cells to fully adhere to the plates before proceeding with the assay. 3.) Run the MTT Assay on the standard curve plates as described in Section V. 4.) See Figures 2 and 3 for representative data. 8
Figures 2 and 3: The effect of the concentration of NIH3T3 cells on absorbance at 570nm. Representative Data. 9
V. MTT ASSAY When preparing plates for running the assay, cell suspensions need to be counted and homogenized to ensure that the correct number is consistently added to each well. Preparation of Reagents: The following two reagents need to be prepared from the supplied components before each use. Refer to Table 2 on page 11 for required volumes. 1.) 10mM Hepes in media, ph 7.4 Prepare a volume of HEPES at 10mM in media sufficient to cover the number of wells the assay will be run on. o Weigh out and transfer HEPES into a clean preferably sterile vial. o Dissolve HEPES in minimal growth media without calf serum. Use the media appropriate for the cell line the assay is being run on. o Adjust the ph to 7.4 with 1.0M NaOH or HCl dropwise. o Keep the media warm at 37 o C until use. 2.) MTT o Weigh and add 2mg MTT powder per ml of MTT Buffer (PBS). Use Table 2 to determine amount to be prepared. Running the Assay: 1.) Remove growth media from the wells of the plates using an aspirator under a laminar flow hood. 2.) Rinse wells 2-3 times with 1X PBS or appropriate isotonic salt buffer, aspirating off each time. 3.) Add prepared media containing 10mM HEPES, ph 7.4 to each well (refer to Table 2 for required volume.) 4.) Add prepared 2 mg/ml MTT in PBS to each well. (Again, refer to Table 2 for volume.) 10
Table. 2: Reagent volumes versus plate type Amount of Media Amount of Plate w/10mm HEPES 2 mg/ml MTT Type per well per well Amount of Solubilization Solution per well 96 well 160 µl 40 µl 100 µl 12 well 0.80 ml 0.20 ml 0.50 ml 6 well 1.60 ml 0.40 ml 1.00 ml 60 mm 4.00 ml 1.00 ml 2.50 ml 100 mm 8.0 ml 2.0 ml 5.0 ml 5.) Swirl plates to mix. 6.) Cover and wrap plates in aluminum foil (to block out light from the plates). 7.) Incubate plates at 37 o C with 5% CO 2 (CO 2 Incubator) for 3 Hours. 8.) After 3 Hours, remove the plates from the incubator and remove the foil. 9.) There will be purple precipitate settled on the bottom of the wells. 10.) Using an aspirator remove the HEPES/MTT Media by aspirating down the side of the well, being careful not to aspirate off any of the precipitate. 11.) Add Solubilization Solution to each well (See Note Below). 12.) Swirl plate until precipitate dissolves (dissolves readily). 13.) For microtiter plates (96, 12, and 6 well plates) the absorbance can be read at 570nm using a plate reader. For 60 and 100mm plates the absorbance needs to be read at 570nm using a spectrophotometer. NOTE: If not able to take reading immediately, DO NOT add Solubilization Solution. Rewrap plates in aluminum foil and store in the freezer. Once the Solubilization Solution is added the reading should be taken as soon as possible. If this is done, all comparative samples need to be frozen, and the standard curve needs to be generated from plates that have been frozen as well to ensure consistency. NOTE: When using a spectrophotometer, solutions with an absorbance value over 0.8 should be diluted with the Solubilization Solution and re-read for a more accurate measurement. Figures would subsequently need to be multiplied by the dilution factor used. NOTE: The Purple formazan produced is somewhat light sensitive, so in all cases plates should be stored in the dark or wrapped in aluminum foil for storage if not read immediately. 11
REFERENCES 1.) Mosmann T. (1983) "Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays." J. Immun. Meth. 65 (1-2): 55 63. 2.) Denizot F, Lang R. (1986). Rapid colorimetric assay for cell growth and survival. Modifications to the tetrazolium dye procedure giving improved sensitivity and readability. J. Immunol. Meth. 89: 271-277. 3.) Carmichael J, DeGraff WG, Gazdar AF, Minna JD, Mitchell JB. (1987) Evaluation of a tetrazolium-based semiautomated colorimetric assay: assessment of radiosensitivity. Cancer Research 47(4): 943-6. 4.) Alley MC, Scudiero DA, Monks A, Hursey ML, Czerwinski MJ, Fine DL, Abbott BJ, Mayo JG, Shoemaker RH, Boyd MR. (1988) Feasibility of drug screening with panels of human tumor cell lines using a microculture tetrazolium assay. Cancer Research 48(3):589-601. 5.) Rubinstein LV, Shoemaker RH, Paull KD, Simon RM, Tosini S, Skehan P, Scudiero DA, Monks A, Boyd MR. (1990) Comparison of in vitro anticancerdrug-screening data generated with a tetrazolium assay versus a protein assay against a diverse panel of human tumor cell lines. Journal of the National Cancer Institute 82(13):1113-18. 6.) Scudiero DA, Shoemaker RH, Paull KD, Monks A, Tierney S, Nofziger TH, Currens MJ, Seniff D, Boyd MR. (1988) Evaluation of a soluble tetrazolium/formazan assay for cell growth and drug sensitivity in culture using human and other tumor cell lines. Cancer Research 48(17): 4827-33. 7.) Gonzalez B, Akman S, Doroshow J, Rivera H, Kaplan WD, Forrest GL. (1995) Protection against daunorubicin cytotoxicity by expression of a cloned human carbonyl reductase cdna in K562 leukemia cells. Cancer Research 55(20): 4646-50. 8.) Marks DC, Belov L, Davey MW, Davey RA, Kidman AD. (1992) The MTT cell viability assay for cytotoxicity testing in multidrug-resistant human leukemic cells. Leukemia Research 16(12): 1165-73. 9.) Bernas T, Dobrucki J. (2002) Mitochondrial and nonmitochondrial reduction of MTT: interaction of MTT with TMRE, JC-1, and NAO mitochondrial fluorescent probes. Cytometry 47(4):236-42. 12
M1475 KIT CONTENTS REAGENTS QUANTITY PART NO. STORAGE MTT 1 x 100mg 1475-001 C, L MTT Buffer (PBS) 1 x 50mL 1475-002 R HEPES 1 x 500mg 1475-003 R Solubilization Solution 1 x 120mL 1475-004 R DOCUMENTATION MSDS Sheets 3 Product Information Sheet 1 Notes: F=store at or below -20 o C; R=store at room temperature; C=store cold (4 o C); L=light sensitive; D=store desiccated; FL=flammable; G=wear protective clothing/gloves/safety glasses when using; B=avoid breathing dust/fumes. 13
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Contact and Support For questions or comments on this or any product from Marker Gene Technologies, Inc., you may contact us by phone or via our website. We welcome customer feedback and we make every effort to improve our products based on input from our clients. To ask a question or make a comment or suggestion, you can call us at 1-888-218-4062 or fax to 541-342-1960. For more information on our products and services, please visit our website at www.markergene.com, where you can find: Secure online ordering Product Information MGT Monthly Newsletters Corporate Information Custom Synthesis Info We want to thank you for your purchase and hope that you will continue to order from Marker Gene Technologies, Inc. 15
Marker Gene Technologies, Inc. University of Oregon Riverfront Research Park 1850 Millrace Drive Eugene, Oregon 97403 1-888-218-4062 www.markergene.com 16