JPH05244993A - Improved method for colorimetric determination of mtt - Google Patents

Abstract

PURPOSE:To solve problems such as much labor and time required for dissolving due to sparing solubility of the formed 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) formazan crystal in carrying out tests on the cell growth ability or cell function according to a method for colorimetric determination of MTT. CONSTITUTION:The objective method for colorimetric determination of MTT is characterized by using a mixture solution of dimethylforamide with isopropyl alcohol containing hydrochloric acid or a mixture solution of dimethyl sulfoxide with the isopropyl alcohol containing the hydrochloric acid for dissolving an MTT formazan crystal. Thereby, the efficient and rapid method for colorimetric determination of the MTT in which the formazan crystal formed by the method for colorimetric determination of the MTT can readily be dissolved without requiring much time is provided.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an improvement in the MTT colorimetric quantification method used for cell growth ability and cell function tests, and more particularly to a method for dissolving produced MTT formazan crystals.

[0002]

2. Description of the Related Art As a method for testing in vitro cell proliferation ability and cell function, a method of measuring uptake of radioisotope (RI) -labeled thymidine ( ³ H-TdR) into cells is generally used. ing. However, since RI is used, there is a risk of being exposed to radiation, and because RI facilities are required, its use is limited.

Therefore, many methods that can be substituted for the ³ H-TdR method have been studied, but the MTT colorimetric method developed by Mosmann (Journal of Immunological Methods 63: 55-63, 1983) is a non-RI method.
It is being used more and more because it is quick and simple. M
In the TT colorimetric method, pale yellow MTT (3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide) selectively acts on mitochondria of living cells, especially its respiratory chain. The enzyme involved in the formation of
It utilizes the fact that TT is cleaved and reduced to produce a dark blue MTT formazan dye. The amount of this formazan can be colorimetrically determined and is proportional to the proliferative ability of living cells. As a method for dissolving the produced MTT formazan dye crystals, 0.04N hydrochloric acid-containing isopropyl alcohol solution is added to the culture solution and pipetting, or 10% sodium dodecylsulfate (SDS) -0.04N hydrochloric acid is added and rapidly mixed. Although the method of leaving it at 37 ° C. overnight is used, there is a problem that the solubility of MTT formazan dye crystals is poor and the former requires time and effort for pipetting and the latter takes time.

[0004]

SUMMARY OF THE INVENTION Therefore, an object of the present invention is to dissolve a MTT formazan crystal, which is produced as described above, when it is tested for cell growth ability and cell function by an MTT colorimetric assay method. It is to provide a means for solving the problem of taking time.

[0005]

[Means for Solving the Problems] The above-mentioned problem is to generate MTT.
Dimethylformamide (DMF) for dissolving formazan crystals
It was solved by using a mixed solution of isopropyl alcohol containing hydrochloric acid or a mixed solution of dimethyl sulfoxide (DMSO) and isopropyl alcohol containing hydrochloric acid.

[0006] In a typical procedure of the conventional MTT colorimetric assay, 100 μl of a cell suspension prepared by using RPMI1640 medium + 10% calf serum is dispensed into each well of a 96-well flat-bottomed microplate and kept for a certain period of time. 37 ° C, 5% C
Place in O ₂ incubator. After that, MTT liquid (MTT 5 mg
Was dissolved in 1 ml of PBS (-) and sterilized by filtration)
10 μl is added, and the mixture is reacted at 37 ° C. for 2 to 6 hours. Next, in order to dissolve the produced MTT formazan crystal,
0.04N Hydrochloric Acid-Containing Isopropyl Alcohol Solution 100μ
Add l and pipet or 10% SDS
After adding 100 μl of −0.04N hydrochloric acid solution, the mixture was quickly mixed and allowed to stand at 37 ° C. overnight, and the amount of MTT formazan was adjusted to 570.
Quantitation is performed by the difference in absorbance between nm and 630 nm.

In the method of the present invention, D is used instead of the isopropyl alcohol solution containing hydrochloric acid or the SDS-hydrochloric acid solution.
A mixed solution of MF and isopropyl alcohol containing hydrochloric acid or a mixed solution of DMSO and isopropyl alcohol containing hydrochloric acid is used. According to the method of the present invention, the MTT formazan crystal is easily and quickly dissolved, the problems of the conventional method are solved, and MTT colorimetric determination can be carried out quickly and easily.

A preferred embodiment of the MTT formazan crystal dissolution method according to the present invention will be described below. The method of the present invention is
The same procedure as the conventional MTT colorimetric method can be performed except that a mixed solution of DMF and isopropyl alcohol containing hydrochloric acid or a mixed solution of DMSO and isopropyl alcohol containing hydrochloric acid is used to dissolve the MTT formazan crystals. it can. Therefore, first, a cell suspension containing test cells and MTT are reacted for 2 to 6 hours in the same manner as in the conventional MTT colorimetric method, and then a mixed solution of DMF and isopropyl alcohol containing hydrochloric acid or DMSO and isopropyl alcohol containing hydrochloric acid is added. The resulting mixture is added, and the resulting crystals are dissolved by lightly irradiating with ultrasonic waves or shaking. At this time, 4
It is preferable to heat to about 0 ° C. Regarding the mixing ratio of the cell suspension and the mixed solution, the higher the ratio of the mixed solution is, the higher the solubility is. However, when the protein is dissolved, when the ratio of the mixed solution is large, the precipitation of the serum protein is caused and the absorbance is affected. Will be given. From these viewpoints, the mixing ratio of the cell suspension and the mixed solution is preferably 1: 2 to 2: 1.

The cell suspension, the mixed solution of DMF and isopropyl alcohol containing hydrochloric acid or the mixed solution of DMSO and isopropyl alcohol containing hydrochloric acid are preferably mixed in equal amounts, in which case the hydrochloric acid content of isopropyl alcohol is 0. It is preferably about 0.04N. The mixing ratio of DMF and isopropyl alcohol containing hydrochloric acid and DMSO and isopropyl alcohol containing hydrochloric acid is from 1: 9 to
It can be 9: 1, particularly preferably 1: 9 to 1: 1 and most preferably 1: 9.

If it is still difficult to lyse, the cell suspension is reacted with MTT and then centrifuged to remove a certain amount of the medium, and then a mixed solution of DMF and isopropyl alcohol containing hydrochloric acid or a mixed solution of DMSO and isopropyl alcohol containing hydrochloric acid. Was added to the DM to dissolve the MTT formazan crystals.
Mixed liquid of F and isopropyl alcohol containing hydrochloric acid or D
The relative amount of the mixed solution of MSO and isopropyl alcohol containing hydrochloric acid can be increased, and the mixture can be heated to about 40 ° C. and irradiated with ultrasonic waves or shaken.

A water bath type ultrasonic wave irradiator can be used for ultrasonic wave irradiation for promoting dissolution, and a microplate mixer can be used for shaking. The MTT colorimetric assay according to the present invention comprises a cell proliferation ability test, culture cell viability measurement, cell function test (complement-related cell lysis test, etc.), measurement of cell-mediated immune response to antigen, drug sensitivity test of anticancer drug, etc. It can be applied to immunosuppression or activator screening. Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited thereto.

[0012]

【Example】

Example 1 Lysis of MTT formazan crystals Human lymphocytes purified from peripheral blood were adjusted to a concentration of 2.0 × 10 ⁶ cells / ml using RPMI1640 medium containing 10% calf serum, and 50 μl each of 96-well microplates. Of each well. Further, 50 μl of PHA (phytohemagglutinin) adjusted to 10 μg / ml was added, and the mixture was cultured at 37 ° C. in a 5% CO ₂ incubator for 72 hours.
After completion of culture, MTT solution (MTT 5 mg was added to 1 ml of PBS).
(10 μl after being sterilized by filtration and sterilized by filtration) was added, and the temperature was 37 ° C.
After reacting for 6 hours, the produced MTT formazan crystals were dissolved in various solvents. The results are shown in the table below. ─────────────────────────────────── Solvent used and dissolution promotion operation Solubility Dissolution time ──── ─────────────────────────────── HCl-IPA, pipetting (conventional method) ○ 20 minutes 10% containing 0.04N hydrochloric acid SDS, overnight standing (conventional method) ○ Overnight 0.04N hydrochloric acid containing 0.3% SDS, overnight standing (conventional method) ○ Overnight HCl-IPA: DMSO = 9: 1, ultrasonic irradiation, heating ◎ 2 minutes HCl-IPA : DMSO = 1: 1, ultrasonic irradiation, heating ○ 2 minutes HCl-IPA: DMF = 9: 1, ultrasonic irradiation, heating ◎ 2 minutes HCl-IPA: DMF = 1: 1, ultrasonic irradiation, heating Temperature ○ 2 minutes ─────────────────────────────────── Solubility ○: OD ₅₆₅ / OD ₆₀₀ = 1.3 more ◎: abbreviations less than _{OD 565 / OD 600 = 1.3 HCl} -IPA: 0.04N hydrochloric acid containing iso B Pill alcohol SDS: sodium dodecyl sulfate DMF: dimethylformamide DMSO: dimethyl sulfoxide Example contains 2 human lymphocyte proliferation measured human lymphocytes by lectin stimulation of 10% calf serum RPMI164
Adjust to 2.0 × 10 ⁶ cells / ml using 0 medium, and
μl was dispensed into each well of a 96-well microplate. Further, PHA 50μ adjusted to 4 to 100 μg / ml
1 was added and the cells were cultured at 37 ° C. in a 5% CO ₂ incubator for 72 hours. After the culture was completed, 10 μl of MTT solution (5 mg of MTT was dissolved in 1 ml of PBS (−) and sterilized by filtration) was added.
After 6 hours, 100 μl of a solvent having a mixing ratio of DMF and isopropyl alcohol containing 0.04 N hydrochloric acid of 1: 9 was added to the produced MTT formazan crystal, and the mixture was heated to about 40 ° C. and lightly irradiated with ultrasonic waves to dissolve. Lymphocyte proliferation was measured by the absorbance of the produced MTT formazan.

The results are shown in FIG. FIG. 1 is a graph in which the difference in absorbance between the added PHA concentration and the produced MTT formazan is plotted. With this method, the optimum concentration of PHA stimulation is 5μ
It was determined to be g / ml. This agrees with the value obtained by the ³ H-TdR method.

[Brief description of drawings]

FIG. 1 Produced MTT measured using the method of the invention in a lectin-stimulated proliferation assay of human lymphocytes.
It is a figure which shows the relationship between the light absorbency difference of formazan, and the addition PHA density | concentration.

Claims (1)

[Claims] 1. An MT characterized by using a mixed solution of dimethylformamide and isopropyl alcohol containing hydrochloric acid or a mixed solution of dimethyl sulfoxide and isopropyl alcohol containing hydrochloric acid to dissolve MTT formazan crystals.
T colorimetric method.