Cell Proliferation Kit I (MTT)
Colorimetric assay (MTT based) for the non-radioactive quantification of cell proliferation and viability
Cat. No. 1 465 0071 Kit (for 2500 tests)
Introduction
The determination of cellular proliferation, viability and activation are key areas in a wide variety of cell biolog-ical approaches. The need for sensitive, quantitative, reliable and automated methods led to the develop-ment of standard assays. Such an example is based on the capability of the cells to incorporate a radioactively labeled substance ([3H])- thymidine), or to release a radioisotope such as [51Cr] after cell lysis. Alternatively, the incorporation of 5-bromo-2’-deoxyuridine (BrdU)* in place of thymidine is monitored as a parameter for DNA synthesis and cellular proliferation in immuno-histo- and cytochemistry, in a cell ELISA and FACS analysis. (kits and reagents for these applications are available from Roche Molecular Biochemicals).
Cell proliferation and viability assays are of particular importance for routine applications. Tetrazolium salts (e.g. MTT, XTT, WST-1) are especially useful for assaying the quantification of viable cells, because they are cleaved to form a formazan dye (fig. 1; for UV absor-bance spectrum, see fig. 2) only by metabolic active cells.
Fig.1: Metabolization of MTT to a formazan salt by viable cells.
1.00.8 ec0.6nabrob0.4a0.20380414448482516550584618652686720wavelength [nm]Fig. 2: Comparison of UV-spectra of MTT labeling reagent (dotted line) and the formazan salt after solubilization with solubilization solution.
0200.C72.2.1465 503R CB GPM
Store at Ϫ15 to Ϫ25°C
The non-radioactive, colorimetric assay system using MTT was first described by Mosmann, T. et al. (1) and improved in subsequent years by several other investi-gators (2–6). The assay is designed for the spectro-photometric quantification of cell growth and viability (1, 3, 5–7) without the use of radioactive isotopes. It is used for the measurement of cell proliferation in response to growth factors, cytokines and nutrients (1–3, 6, 8–12) (see fig. 4).
The MTT assay is also useful for the measurement of cytotoxicity. Examples are the quantification of tumor necrosis factor-a or -b effects (13, 14). (see fig. 5) or macrophage induced cell death (15, 16) and the assessment of cytotoxic (17–34) or growth inhibiting agents such as inhibitory antibodies (see fig. 6).
For the replacement of the radioactive [51Cr]-release cytotoxicity assay, protocols using MTT have been developed. The MTT assay is as sensitive as the radio-active method, but shows a significantly lower back-ground especially after long term incubation (34). The MTT assay can also be used to study cell activation (4). Compared to radioactive isotope techniques, the Cell Proliferation Kit I (MTT) is•safer-no radioactive isotopes are used,•accurate-the absorbance revealed, strongly
correlates to the cell number, (see fig. 3)
•sensitive-low cell numbers are detected (see fig. 3).•fast-the use of multiwell-ELISA readers
allows for processing a large number of samples,
•easy-no washing steps and no additional
reagents are required,
Product description
The kit contains
1. MTT labeling reagent, (1×, ready-to-use)
5 vials containing 5 ml MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromid) labeling reagent (1x), 5 mg/ml, in phosphate buffered saline (PBS), non-sterile, ready to use.
Stability
Stable at Ϫ15 to Ϫ25°C protected from light. Repeated thaw-freeze cycles do not affect product stability. After thawing, the MTT labeling reagents may be stored pro-tected from light at 2-8°C for up to 4 weeks, in which case a sterile filtration of the reagent is recommended.
2. Solubilization solution, (1×, ready-to-use)
3 bottles solubilization solution (90 ml per bottle), 10% SDS in 0.01 M HCI.
Stability
Stable at 15-25°C.
Note
Application
Assay principle
Precipitates may form during shipment or storage, in Assay procedure
which case the container should be warmed to 37°C and thoroughly mixed.
1. Preparation of solutions
1.50All solutions are ready to use. (No additional reagents are required.)
1.252.Working instruction
]mn•Cells are grown in microtiter plates (tissue culture 091.00grade, 96 wells, flat bottom) in a final volume of 6A100 l culture medium per well, according to the –mmedia needs of the cells, in a humidified atmosphere n 0(e.g. 37°C, 6.5% CO2). The incubation period of the 50.755cell cultures depends on the particular experimental A[approach and on the cell line used for the assay. For ecmost experimental setups, the incubation of cells for na0.5024 to 96 h is appropriate.
bro•After the incubation period, add 10 l of the MTT balabeling reagent (final concentration 0.5 mg/ml) to 0.25each well.
•Incubate the microtiter plate for 4 h in a humidified atmosphere (e.g. 37°C, 6.5% CO2).
0.00•Add 100 l of the solubilization solution into each 103104105106well.
cellnumber(cells/ml)•Allow the plate to stand overnight in the incubator in a humidified atmosphere (e.g. 37°C, 6.5% CO2). Fig. 3: Effect of different numbers of cells on color formation •Check for complete solubilization of the purple for-(example given, using Ag8 cells).
mazan crystals and measure the spectrophotometri-cal absorbance of the samples using a microtiter plate (ELISA) reader. The wavelength to measure absorbance of the formazan product is between 550 and 600 nm according to the filters available for the ELISA reader, used. The reference wavelength MTT is used for the quantitative determination of
should be more than 650 nm.
cellular proliferation and activation e.g. in response to growth factors and cytokines such as IL-2 and IL-6 Note: If for the initial incubation of the cells a larger (1-3, 6, 8-11). It is also used for the quantification of volume of culture medium is required, increase the antiproliferative or cytotoxic effects e.g. mediated by amount of MTT labeling reagent correspondingly (e.g. tumor necrosis factor-α or -β (13, 14) and for the 20 l MTT labeling reagent, when cells are cultured in measurement of interferon action (12). In cancer 200 l culture medium).
research the MTT assay is used for quantification of in vitro chemosensitivity of tumor cells (16-31) and for the screening of anticancer compounds (32, 33). MTT, when dissolved in the agarose overlay is also employed Examples
to improve the visualization of virus infected cells in the plaque forming assay (34).
1. Cell growth assay procedure
The assay is based on the cleavage of the yellow tetra-zolium salt MTT to purple formazan crystals by meta-To determine the activity of human interleukin-6 (hIL-6) bolic active cells (fig. 1) (6, 7, 35). This cellular reduction on 7TD1 cells (mouse-mouse hybridoma) (see fig. 4)
involves the pyridine nucleotide cofactors NADH and NADPH (36). The formazan crystals formed are solubi-0.9lized and the resulting colored solution is quantified using a scanning multiwell spectrophotometer (ELISA 0.8reader). This ensures a high degree of accurracy,
enables on-line computer processing of the data (data ]m0.7collection, calculation and report generation) and, n 0thereby, allows the rapid and convenient handling of a 96high number of samples.
A0.6–mCells, grown in a 96 well tissue culture plate, are incu-n0.5 0bated with the yellow MTT solution (final concentration 550.5 mg/ml) for approx. 4 h. After this incubation period, A0.4[ purple formazan salt crystals are formed. These salt eccrystals are insoluble in aqueous solution, but may be n0.3asolubilized by adding the solubilization solution and brincubating the plates overnight in humidified atmo-ob0.2asphere (e.g. 37°C, 6.5% COproduct is spectrophotometrically quantified using an 2). The solubilized formazan 0.1ELISA reader. An increase in number of living cells results in an increase in the total metabolic activity in 0.00.11101001000the sample. This increase directly correlates to the hIL-6[pg/ml]amount of purple formazan crystals formed, as monitored by the absorbance (see fig. 3).
Fig. 4: Proliferation of 7TD1 cells (mouse-mouse hybridoma) in response to recombinant human interleukin-6 (hIL-6) using the procedure described (see section Examples, 1.).
2
Roche Molecular Biochemicals
Reagents
•Culture medium, e.g. DMEM containing 10% heat inactivated FCS (fetal calf serum), 2 mM glutamine, 3. Assay procedure for the analysis of neutralizing monoclonal antibodies to growth factors or cytokines
0.55 mM L-arginine, 0.24 mM L-asparagine-mono-hydrate, 50 M 2-mercaptoethanol, HT-media sup-plement* (1×), containing 0.1 mM hypoxanthine and 16 M thymidine. If an antibiotic is to be used, addi-tionally supplement media with penicillin/streptomy-cin* or gentamicin*.
•Interleukin-6, human (hIL-6) (200 000 U/ml, 2 g/ml)*, sterile.
Reagents:
•Cell Proliferation Kit I (MTT).
Procedure
•Seed 7TD1 cells at a concentration of 2 × 103 cells/well in 100 l culture medium containing various amounts of IL-6 [final concentration e.g. 0.1–10 U/ml (0.001–0.1 ng/ml)] into microtiter plates (tissue culture grade, 96 wells, flat bottom). Incubate cell cultures for 4 days at 37°C and 6.5% CO2.
•Add 10 l MTT labeling solution and continue as described under working instruction (section Assay procedure, 2.). 2. Cytotoxicity assay procedure •To determine the cytotoxic effect of human tumor necrosis factor-␣ (hTNF-␣) on WEHI-164 cells (mouse fibrosarcoma) (see fig. 5). Reagents
•Culture medium, e.g. RPMI 1640 containing 10% heat inactivated FCS (fetal calf serum), 2 mM glutamine and 1g/ml actinomycin C (actinomycin D)*. If an antibiotic is to be used, additionally supplement media with penicillin/streptomycin* or gentamicin*. •Tumor necrosis factor-α, human (hTNF-α) (10 g/ml)*, sterile. •Cell Proliferation Kit I (MTT). 0.5]0.4mn 096A–0.3mn 055A[ e0.2Procedurecnabroba0.10.00.010.11101001000hTNF-␣[pg/ml]Fig. 5: Determination of the cytotoxic activity of recombinant human Note
TNF-␣ (h TNF-␣) on WEHI-164 cells (mouse fibrosarcoma) using the procedure described (see section Examples, 2.).
Procedure
•Preincubate WEHI-164 cells at a concentration of 1 × 106 cells/ml in culture medium with 1 g/ml actinomycin C1 for 3 h at 37°C and 6.5% CO42.
•Seed cells at a concentration of 5 × 10 cells/well in 100 l culture medium containing 1 g/ml actino-mycin Ccentration e.g. 0.0011 and various amounts of hTNF-α (final con-–0.5 ng/ml) into microtiter plates (tissue culture grade, 96 wells, flat bottom). Incubate cell cultures for 24 h at 37°C and 6.5% CO2. •Add 10 l MTT labeling solution and continue as described under working instruction (section Assay procedure, 2).
3
To determine the inhibitory activity of a murine, mono-clonal antibody to human granulocyte-macrophage colony stimulating factor (anti-hGM-CSF) on hGM-CSF activity on TF-1 cells (human erythroleukemic cells) (see fig. 6).
•Culture medium, e.g. RPMI 1640 containing heat inactivated 10% FCS (fetal calf serum), 2 mM L-glutamine. If an antibiotic is to be used, additionally supplement media with penicillin/streptomycin* or gentamicin*.
•hGM-CSF (10 000 U/ml, 1 g/ml)*, sterile.
•anti-hGM-CSF (200 g/vial)*, lyophilized, sterile. •Cell Proliferation Kit I (MTT).
0.7]m0.6n 096A–mn0.5 055A[ ecn0.4abroba0.30.20.010.1110100Anti-hGM-CSF[g/ml]Fig. 6: Inhibition of recombinant human GM-CSF (5 U/ml; 0.1 ng/ml) (᭹), but not recombinant human interleukin-3(0.4 U/ml; 0.2 ng/ml) (᭺) activity on TF-1 cells (human erythro-leukemic cells) by anti-hGM-CSF (clone 3092) using the proce-dure described (see section Examples, 3.). •Preincubate culture medium containing hGM-CSF (5 U/ml, 0.1 ng/ml) and various amounts of anti-hGM-CSF (final concentration e.g. 0.01–50 g/ml) in microtiter plates (tissue culture grade, 96 wells, flat bottom) •Add TF-1 cells at a concentration of 5 × 103 cells/well in 50 l culture medium and incubate for 48 h. •Add 10 l MTT labeling solution and continue as described under working instruction (section Assay procedure, 2.). Recombinant, human interleukin-3 (hIL-3)*, which also is effective on TF-1 cells, can be used as a negative control (see fig. 6).
Roche Molecular Biochemicals
Cell Proliferation Kit II (XTT) flow chartStepProcedure
Vol./well
TimeTemp. (°C)Perform tissue culture using 100l
24-96 h
37°C
96 well microtiter plates
(tissue culture grade, flat-bottom)1
Add MTT labeling reagent 10 l4 h37°C
and incubate in a humidified atmosphere
2Add solubilization solution 100 lovernight37°C
and incubate in a humidified atmosphere
3Evaluate microtiter plate with ___
the use of an ELISA reader at 550–600 nm with a reference wavelength of Ͼ 650 nm
Note: If for the initial incubation of the cells a larger volume of culture medium is required, increase the amount of MTT labeling reagent correspondingly (e.g. 20 l MTT labeling reagent, when cells are cultured in 200 l culture medium).
References
1Mosmann, T. et al. (1983) J. Immunol. Methods 65, 55–63. 2Tada, H. et al. (1986) J. Immunol. Methods 93, 157–165.
3Denizot, F. & Lang, R. (1986) J. Immunol. Methods 89, 271–277. 4Gerlier, D. & Thomasset, N. (1986) J. Immunol. Methods 94, 57–63.
5Hansen, M. B., Nielsen, S. E. & Berg, K. (1989) J. Immunol. Methods 119, 203–210.6Vistica, D. T. et al. (1991) Cancer Res. 51, 2515–2520.
7Maehara, Y. et al. (1986) Eur. J. Cancer Clin. Oncol. 23, 273–276. 8Heeg, K. et al. (1985) J. Immunol. Methods 77, 237–246.
9Hooton, J. W. L., Gibbs, C. & Paetkan, U. (1985) J. Immunol. 135, 2464–2473. 10Mosmann, T. R. & Fong, T. A. T. (1989) J. Immunol. Methods 116, 151–158. 11Ohno, M. & Abe, T. (1991) J. Immunol. Methods 145, 199–203.
12Berg, K., Hansen, M. B. & Nielsen, S. E. (1988) J. Interferon Res. 8, (suppl. 1), S. 67. 13Green, L. M., Reade, J. L. & Ware, C. F. (1984) J. Immunol. Methods 70, 257–268.14Espevik, T. & Nissen-Meyer, J. (1986) J. Immunol. Methods 95, 99–105.
15Ferrari, M., Fornasiero, M. C. & Isetta, A. M. (1990) J. Immunol. Methods 131, 165–172. 16Van de Loosdrecht, A. A. et al. (1991) J. Immunol. Methods 141, 15–22. 17Cole, S. P. C. (1986) Cancer Chemother. Pharmacol 17, 259–263. 18Carmichael, J. et al. (1987) Cancer Res. 47, 936–942.
19Twentyman, P. R. & Luscombe, M. (1987) Br. J. Cancer 56, 279–285. 20Park, J.-G. et al. (1987) Cancer Res. 47, 5875–5879.
21Horiuchi, N. et al. (1988) Cancer Chemother. Pharmacol. 22, 246–250. 22Pieters, R. et al. (1988) Cancer Letters 41, 323–332.
23McHale, A. P. & McHale, L. (1988) Cancer Letters 41, 315–321.
24Edmondson, J. M., Armstrong, L. S. & Martinez, A. O. (1988) J. Tissue Culture Methods 11, 15–17.
25Faircloth, G. T., Stewart, D. & Clement, J. J. (1988) J. Tissue Culture Methods 11, 201–205. 26Campling, B. G. et al. (1988) Leukemia Res. 12, 823–831. 27Pieters, P. et al. (1989) Br. J. Cancer 59, 217–220.
28Twentyman, P. R., Fox, N. E. & Rees, K. H. (1989) Br. J. Haematol. 71, 19–24. 29Plumb, J. A., Milroy, R. & Kaye, S. B. (1989) Cancer Res. 49, 4435–4440. 30Alley, M. C. et al. (1988) Cancer Res. 48, 589–601. 31Heo, D. S. et al. (1990) Cancer Res. 50, 3681–3690.
32Ruben, R. L. & Neubauer, R. H. (1987) Cancer Treat. Rep. 71, 1141–1149. 33Rubinstein, L. V. et al. (1990) J. Natl. Canc. Inst. 82, 1113–1118. 34Shanafelt, A. B. (1991) BioTechniques 3, 330.
35Slater, T. F., Sawyer, B. & Sträuli, U. (1963) Biochim.
36
Berridge, M. V. & Tan, A. S. (1993) Arch. Biochem. Biophys. 303, 474.
Note: A reference list for microtiter tetrazolium assays (e.g. MTT, XTT, WST-1)
is available on request.* available from Roche Molecular Biochemicals
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