MTT Cell Growth Kit

Key Applications
ACT

Key Applications

ACT

Description
Catalogue Number	CT02
Brand Family	Chemicon®
Trade Name	Chemicon
Description	MTT Cell Growth Assay Kit
Overview	MTT is a pale yellow substrate that is cleaved by living cells to yield a dark blue formazan product. This process requires active mitochondria, and even freshly dead cells do not cleave significant amounts of MTT. The colorimetric assay described below can be used for either proliferation or complement-mediated cytotoxicity assays.
Materials Required but Not Delivered	Isopropanol with 0.04 N HCl for color development

Description

Catalogue Number

CT02

Brand Family

Chemicon®

Trade Name

Chemicon

Description

MTT Cell Growth Assay Kit

Overview

MTT is a pale yellow substrate that is cleaved by living cells to yield a dark blue formazan product. This process requires active mitochondria, and even freshly dead cells do not cleave significant amounts of MTT. The colorimetric assay described below can be used for either proliferation or complement-mediated cytotoxicity assays.

Materials Required but Not Delivered

Isopropanol with 0.04 N HCl for color development

Product Information
Components	Reagent A: MTT, (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide), 50 mg/vial. Solution B: PBS pH 7.4, 15 mL

Product Information

Components

Reagent A: MTT, (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide), 50 mg/vial.
Solution B: PBS pH 7.4, 15 mL

Applications
Application	MTT Cell Growth Assay is a colorimetric assay that can be used for either proliferation or complement-mediated cytotoxicity assays.
Key Applications	Activity Assay
Application Notes	Procedure: 1) Carry out a lymphokine, mitogen, or complement-mediated cytotoxicity assay using standard methods, in 96-well flat-bottomed tissue culture plates of good optical quality (e.g. Falcon). The final volume of tissue culture medium in each well should be 0.1 mL, and the medium (e.g. RPMI or DMEM) may contain up to 10% Fetal Bovine Serum. 2) At the end of the assay add 0.01 mL AB Solution (MTT) to each well. Mix by tapping gently on the side of the tray. 3) Incubate at 37° C for cleavage of MTT to occur. Optimal times may vary according to the assay, but four hours is suitable for most purposes. At the end of this time, the MTT formazan produced in wells containing live cells will appear as black, fuzzy crystals on the bottom of the well. 4) Add 0.1 mL isopropanol with 0.04 N HCl to each well. Mix thoroughly by repeated pipetting with a multichannel pipettor. The HCl converts the phenol red in tissue culture medium to a yellow color that does not interfere with MTT formazan measurement. The isopropanol dissolves the formazan to give a homogeneous blue solution suitable for absorbance measurement. 5) Within an hour, measure the absorbance on an ELISA plate reader with a test wavelength of 570 nm and a reference wavelength of 630 nm. After several hours at room temperature, serum proteins may begin to precipitate due to the acid/alcohol. Chilling the plates will hasten the precipitation. If the plates must be stored before measuring, keep at 4° C before adding the acid/alcohol, then warm to room temperature and add acid/alcohol just before reading. Results: The MTT assay will normally detect 200 to 50,000 cells of a typical cell line, although 1,000 to 50,000 is the useful range. This number may vary for other cell types. Cytotoxic assays should be set up so that the control, unlysed cells give a signal of 0.2 to 0.4, and proliferation assays should yield a similar value at plateau concentrations. This corresponds to about 20-50,000 cells per well with a typical cell line. Absorbance is directly proportional to the number of cells; actual cells do not absorb significantly, even up to concentrations of 1 x 10⁶ cells/mL.

Applications

Application

MTT Cell Growth Assay is a colorimetric assay that can be used for either proliferation or complement-mediated cytotoxicity assays.

Key Applications

Activity Assay

Application Notes

Procedure:
1) Carry out a lymphokine, mitogen, or complement-mediated cytotoxicity assay using standard methods, in 96-well flat-bottomed tissue culture plates of good optical quality (e.g. Falcon). The final volume of tissue culture medium in each well should be 0.1 mL, and the medium (e.g. RPMI or DMEM) may contain up to 10% Fetal Bovine Serum.

2) At the end of the assay add 0.01 mL AB Solution (MTT) to each well. Mix by tapping gently on the side of the tray.

3) Incubate at 37° C for cleavage of MTT to occur. Optimal times may vary according to the assay, but four hours is suitable for most purposes. At the end of this time, the MTT formazan produced in wells containing live cells will appear as black, fuzzy crystals on the bottom of the well.

4) Add 0.1 mL isopropanol with 0.04 N HCl to each well. Mix thoroughly by repeated pipetting with a multichannel pipettor. The HCl converts the phenol red in tissue culture medium to a yellow color that does not interfere with MTT formazan measurement. The isopropanol dissolves the formazan to give a homogeneous blue solution suitable for absorbance measurement.

5) Within an hour, measure the absorbance on an ELISA plate reader with a test wavelength of 570 nm and a reference wavelength of 630 nm. After several hours at room temperature, serum proteins may begin to precipitate due to the acid/alcohol. Chilling the plates will hasten the precipitation. If the plates must be stored before measuring, keep at 4° C before adding the acid/alcohol, then warm to room temperature and add acid/alcohol just before reading.

Results:
The MTT assay will normally detect 200 to 50,000 cells of a typical cell line, although 1,000 to 50,000 is the useful range. This number may vary for other cell types. Cytotoxic assays should be set up so that the control, unlysed cells give a signal of 0.2 to 0.4, and proliferation assays should yield a similar value at plateau concentrations. This corresponds to about 20-50,000 cells per well with a typical cell line.

Absorbance is directly proportional to the number of cells; actual cells do not absorb significantly, even up to concentrations of 1 x 10⁶ cells/mL.

Biological Information
Species Reactivity	Vertebrates

Biological Information

Species Reactivity

Vertebrates

Product Usage Statements
Usage Statement	Unless otherwise stated in our catalog or other company documentation accompanying the product(s), our products are intended for research use only and are not to be used for any other purpose, which includes but is not limited to, unauthorized commercial uses, in vitro diagnostic uses, ex vivo or in vivo therapeutic uses or any type of consumption or application to humans or animals.

Product Usage Statements

Usage Statement

Unless otherwise stated in our catalog or other company documentation accompanying the product(s), our products are intended for research use only and are not to be used for any other purpose, which includes but is not limited to, unauthorized commercial uses, in vitro diagnostic uses, ex vivo or in vivo therapeutic uses or any type of consumption or application to humans or animals.

Storage and Shipping Information
Storage Conditions	Maintain at 2-8°C for up to six months. The Reagent A / Solution B mixture is stable at 2-8°C for up to two weeks. Reagent Preparation: Add 10 mL Solution B to one vial of Reagent A. Mix well, sterile filter and keep in the dark at 4° C until used. Note: It may take overnight to dissolve. Do not heat solution. When absolutely required to dissolve crystals, adjust pH with 1-2 drops of HCl. The AB mixture is stable for several weeks under these conditions.

Storage and Shipping Information

Storage Conditions

Maintain at 2-8°C for up to six months. The Reagent A / Solution B mixture is stable at 2-8°C for up to two weeks.

Reagent Preparation:
Add 10 mL Solution B to one vial of Reagent A. Mix well, sterile filter and keep in the dark at 4° C until used. Note: It may take overnight to dissolve. Do not heat solution. When absolutely required to dissolve crystals, adjust pH with 1-2 drops of HCl. The AB mixture is stable for several weeks under these conditions.

Packaging Information
Material Size	1000 assays

Packaging Information

Material Size

1000 assays

实验耗材

分子生物学试剂

蛋白与免疫学

生化试剂

细胞及检测试剂

色谱及层析

仪器设备

实验服务

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