Professional Documents
Culture Documents
1.50
0.8 1.00
aborbance
0.6
0.75
0.4
0.50
0.2
0 0.25
380 414 448 482 516 550 584 618 652 686 720
wavelength [nm]
0.00
Fig. 2: Comparison of UV-spectra of MTT labeling reagent (dotted
line) and the formazan salt after solubilization with solubilization 103 104 105 106
solution. cell number (cells/ml)
Fig. 3: Effect of different numbers of cells on color formation (exam-
ple given, using Ag8 cells).
0316.11465 5030019
sigma-aldrich.com
Application The non-radioactive, colorimetric assay system using Handling instruc- If for the initial incubation of the cells a larger volume
MTT was first described by Mosmann, T. et al. (1) and tion for larger of culture medium is required, increase the amount of
improved in subsequent years by several other investi- volumes MTT labeling reagent correspondingly (e.g., 20 μl MTT
gators (2–6). labeling reagent, when cells are cultured in 200 μl
The assay is designed for the spectrophotometric culture medium).
quantification of cell growth and viability (1, 3, 5–7)
without the use of radioactive isotopes. Protocol Please refer to the following table.
• It is used for the measurement of cell proliferation in Note: If for the initial incubation of the cells a larger
response to growth factors, cytokines and nutrients volume of culture medium is required, increase then
(1–3, 6, 8–12) (see fig. 4). amount of MTT labeling reagent correspondingly (e.g.,
20 μl MTT labeling reagent, when cells are cultured in
• The MTT assay is also useful for the measurement of 200 μl culture medium).
cytotoxicity. Examples are the quantification of
Step Action
tumor necrosis factor-a or -b effects (13, 14). (see
fig. 5) or macrophage induced cell death (15, 16) 1 Cells are grown in microplates (tissue culture
and the assessment of cytotoxic (17–34) or growth grade, 96 wells, flat bottom) in a final volume of
inhibiting agents such as inhibitory antibodies (see 100 μl culture medium per well, according to the
fig. 6). media needs of the cells, in a humidified atmo-
sphere (e.g., +37°C, 5 - 6.5% CO2). The incuba-
• For the replacement of the radioactive [51 Cr]-release tion period of the cell cultures depends on the
cytotoxicity assay, protocols using MTT have been particular experimental approach and on the cell
developed. The MTT assay is as sensitive as the line used for the assay. For most experimental
radioactive method, but shows a significantly lower setups, the incubation of cells for 24 to 96 h is
background especially after long term incubation appropriate.
(34). 2 After the incubation period, add 10 μl of the MTT
• The MTT assay can also be used to study cell activa- labeling reagent (final concentration 0.5 mg/ml)
tion (4). to each well.
3 Incubate the microplate for 4 h in a humidified
Storage and Stable at -15 to -25°C until the expiration date printed atmosphere (e.g., +37°C, 5 - 6.5% CO2 ).
stability on the label.
4 Add 100 μl of the Solubilization solution into
Note: Protect from light. Repeated thaw-freeze cycles each well.
do not affect product stability. Precipitates may form 5 Allow the plate to stand overnight in the incuba-
during shipment or storage, in which case the con- tor in a humidified atmosphere (e.g., +37°C, 5 -
tainer should be warmed to +37°C and thoroughly 6.5% CO2 ).
mixed.
6 Check for complete solubilization of the purple
After thawing, the MTT labeling reagents may be formazan crystals and measure the spectropho-
stored protected from light at +2 to +8°C for up to tometrical absorbance of the samples using a
4 weeks, in which case a sterile filtration of the reagent microplate (ELISA) reader. The wavelength to
is recommended. measure absorbance of the formazan product is
between 550 and 600 nm according to the filters
Advantages Compared to radioactive isotope techniques, the Cell available for the ELISA reader, used. The refe-
Proliferation Kit I (MTT) shows the following benefits. rence wavelength should be more than 650 nm.
Benefit Feature
Safe No radioactive isotopes are used. 3.2 Examples
Accurate The absorbance revealed, 3.2.1. Cell growth assay procedure
strongly correlates to the cell
number, (see fig. 3). Additional • Culture medium, e.g., DMEM containing 10% heat
Sensitive Low cell numbers are detected reagents required inactivated FCS (fetal calf serum), 2 mM glutamine,
(see fig. 3). 0.55 mM L-arginine, 0.24 mM L-asparagine-mono-
Fast The use of multiwell-ELISA read- hydrate, 50 μM 2-mercaptoethanol, HT-media sup-
ers allows for processing a large plement (1×), containing 0.1 mM hypoxanthine and
number of samples. 16 μM thymidine. If an antibiotic is to be used, addi-
Easy No washing steps and no addi- tionally supplement media with penicillin/strepto-
tional reagents are required. mycin or gentamicin *
• Interleukin-6, human (hIL-6) (200,000 U/ml, 2 μg/ml)
sterile*.
3. Protocols and required material Protocol For the determination of human interleukin-6 (hIL-6) activ-
ity on 7TD1 cells (mouse-mouse hybridoma) (see fig. 4).
3.1 Assay procedure
Step Action
Overview Please refer to the following table. 1 Seed 7TD1 cells at a concentration of 2 × 103
cells/well in 100 μl culture medium containing
Step Description Volume/ Time/ various amounts of IL-6 [final concentration e.g.,
well Temp 0.1–10 U/ml (0.001–0.1 ng/ml)] into microplates
Perform tissue culture 100 μl 24-96 h (tissue culture grade, 96 wells, flat bottom).
using 96 well micro- +37°C. 2 Incubate cell cultures for 4 days at +37°C and 5
plates (tissue culture - 6.5% CO2.
grade, flat-bottom)
3 After the incubation period, add 10 μl of the MTT
1 Add MTT labeling 10 μl 4 h +37°C. labeling reagent (final concentration 0.5 mg/ml)
reagent and incubate to each well.
in a humidified atmo-
sphere 4 Incubate the microplate for 4 h in a humidified
atmosphere (e.g., +37°C, 5 - 6.5% CO2).
2 Add solubilization 100 μl overnight 5 Add 100 μl of the Solubilization solution into
solution and incubate +37°C each well.
in a humidified atmo-
sphere 6 Allow the plate to stand overnight in the incuba-
tor in a humidified atmosphere (e.g., +37°C, 5 -
3 Evaluate microplate 6.5% CO2 ).
with the use of an
ELISA reader at 550– 7 Check for complete solubilization of the purple
600 nm with a refer- formazan crystals and measure the spectropho-
ence wavelength of tometrical absorbance of the samples using a
>650 nm. microplate (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 refe-
rence wavelength should be more than 650 nm.
2
sigma-aldrich.com
0.9
0.8 0.5
0.7
0.3
0.2
0.2
0.1
0.1
0.0
0.1 1 10 100 1000
hIL-6 [pg/ml]
0.0
0.01 0.1 1 10 100 1000
Fig. 4: Proliferation of 7TD1 cells (mouse-mouse hybridoma) in
hTNF-␣ [pg/ml]
response to recombinant human interleukin-6 (hIL-6) using the
procedure described (see section Examples, 3.2.1.).
Fig. 5: Determination of the cytotoxic activity of recombinant human
TNF-␣ (h TNF-␣) on WEHI-164 cells (mouse fibrosarcoma) using the
procedure described (see section Examples,3.2.2.).
3.2.2 Cytotoxicity assay procedure
Additional • Culture medium, e.g., RPMI 1640 containing 3.2.3. Assay procedure for the analysis of neutralizing monoclonal
reagents required 10% heat inactivated FCS (fetal calf serum), 2 mM antibodies to growth factors or cytokines
glutamine and 1μg/ml actinomycin C1 (actinomycin
D). If an antibiotic is to be used, additionally supple-
ment media with penicillin/streptomycin or genta- Additional • Culture medium, e.g., RPMI 1640 containing heat
micin* reagents required inactivated 10% FCS (fetal calf serum), 2 mM L-glu-
• Tumor necrosis factor-α, human (hTNF-α) tamine. If an antibiotic is to be used, additionally
(10 μg/ml)*, sterile*. supplement media with penicillin/streptomycin or
gentamicin*.
• hGM-CSF (10,000 U/ml, 1 μg/ml), sterile *
Protocol For the determination of the cytotoxic effect of human
tumor necrosis factor-α (hTNF-α) on WEHI-164 cells • anti-hGM-CSF (200 μg/vial), lyophilized, sterile.
(mouse fibrosarcoma) (see fig. 5).
Protocol For the determination of the inhibitory activity of a
Step Action
murine, monoclonal antibody to human granulocyte-
1 Preincubate WEHI-164 cells at a concentration macrophage colony stimulating factor (anti-hGM-CSF)
of 1 × 106 cells/ml in culture medium with 1 μg/ on hGM-CSF activity on TF-1 cells (human erythro-
ml actinomycin C1 for 3 h at +37°C and 5 - 6.5% leukemic cells).
CO2.
Note: Recombinant, human interleukin-3 (hIL-3)*,
2 Seed cells at a concentration of 5 × 104 cells/ which also is effective on TF-1 cells, can be used as a
well in 100 μl culture medium containing negative control (see fig. 6).
1 μg/ml actinomycin C1 and various amounts of
hTNF-α (final concentration e.g., 0.001–0.5 Step Action
ng/ml) into microplates (tissue culture grade, 1 Preincubate culture medium containing hGM-
96 wells, flat bottom). CSF (5 U/ml, 0.1 ng/ml) and various amounts of
3 Incubate cell cultures for 24 h at +37°C and 5 - anti-hGM-CSF (final concentration e.g., 0.01–
6.5% CO2 . 50 μg/ml) in microplates (tissue culture grade,
96 wells, flat bottom).
4 After the incubation period, add 10 μl of the MTT
labeling reagent (final concentration 0.5 mg/ml) 2 Add TF-1 cells at a concentration of 5 × 103
to each well. cells/well in 50 μl culture medium and incubate
for 48 h.
5 Incubate the microplate for 4 h in a humidified
atmosphere (e.g., +37°C, 5 - 6.5% CO2 ). 3 After the incubation period, add 10 μl of the MTT
labeling reagent (final concentration 0.5 mg/ml)
6 Add 100 μl of the Solubilization solution into to each well.
each well.
4 Incubate the microplate for 4 h in a humidified
7 Allow the plate to stand overnight in the incuba- atmosphere (e.g., +37°C, 5 - 6.5% CO2 ).
tor in a humidified atmosphere (e.g., +37°C, 5 -
6.5% CO2 ). 5 Add 100 μl of the Solubilization solution into
each well.
8 Check for complete solubilization of the purple 6 Allow the plate to stand overnight in the incuba-
formazan crystals and measure the spectropho-
tometrical absorbance of the samples using a tor in a humidified atmosphere (e.g., +37°C, 5 -
microplate (ELISA) reader. The wavelength to 6.5% CO2 ).
measure absorbance of the formazan product is 7 Check for complete solubilization of the purple
between 550 and 600 nm according to the filters formazan crystals and measure the spectropho-
available for the ELISA reader, used. The refer- tometrical absorbance of the samples using a
ence wavelength should be more than 650 nm. microplate (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 refe-
rence wavelength should be more than 650 nm.
3
sigma-aldrich.com
Related Products
0.7