MTT Cell Proliferation Assay sunpingli

Introduction A simple assay to determine the viability and number of cells in culture through the formation of a colored product Yellow MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, a tetrazole salt) is reduced(환원) to blue/purple(보라색) formazan by intracellular NADPH-oxidoreductases (탈수소효소) which present in the mitochondria of living cells.

원리

Introduction The formazan cannot pass through the plasma membrane and accumulates within the cell , the intracellular purple formazan can be solubilized by Dimethylsulfoxide (DMSO) and the concentration can be determined by optical density (OD:흡광도) at 570 nm use the Spectrophotometer(분광광도계). The absorption is directly proportional to the cell number which allows an accurate quantification of cell proliferation, viability, and cytotoxicity assays.

APPLICATIONS Cell proliferation and activation assay Examples:in response to growth factors and cytokines. Cytotoxicity Assay Examples: quantification of tumor necrosis factor-α or -β effects. Drug action, cytotoxic agents and screening other biologically active compounds.

Cytotoxicity Assay Determination of the cytotoxic activity of recombinant human TNF-α (h TNF-α) on WEHI-164 cells (mouse fibrosarcoma) using the procedure described

Dose-response and Time-course effect of growth factors Dose-response effect of growth factors on proliferation of human NP cells . Cells were incubated with increasing concentrations (0.1–500 μg/L) of TGF-β1 and IGF-I in medium for 72 hours. The proliferative response was evaluated using MTT assay. The proliferative response was evaluated by MTT assay at 1-, 3-.5- and 7-day. Results were expressed as the mean optical density of MTT absorbance in growth factors .

Method 1.Plate cells into 96-well tissue culture plates. In general, 5000 - 10,000 cells per well according to the experimental factors tested. 2.Carry out your experiment by adding chemicals or biological agents into appropriate well. Incubate for 6 to 24 hours 3 .Add 10 μl MTT Reagent. 4 .Incubate for 2 to 4 hours until purple precipitate is visible. 5 . Remove medium and add 100μL DMSO into each well to dissolve the formazan. 6 .Leave at room temperature in the dark for 2 hours or Shaking 15min. 7. Record absorbance at 570 nm.

Key features Limitations Safer -no radioactive isotopes(동위원소) are used. Accurate -the absorbance revealed, strongly correlates to the cell number. Sensitive -low cell numbers are detected. 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 Limitations Influenced by: (1) the physiological state of cells and (2) variance in mitochondrial dehydrogenase (산화 환원 효소) activity.

Cell Counting Kit-8 (CCK-8) WSTs Dojindo company developed highly water-soluble tetrazolium salts called WSTs. WSTs receive two electrons from viable cells to generate a yellow, orange, or purple water-soluble formazans. WST-8 A highly stable WST, is utilized in Cell Counting Kit-8 (CCK-8). The electron mediator used in this kit, 1-Methoxy PMS, is also highly stable. WST-8, WST-8 formazan, and 1-Methoxy PMS have no cytotoxicity in cell culture media, additional experiments may be carried out using the same assay plate.

Method 1.Inoculate cell suspension (100 μl/well) in a 96-well plate. 2.Carry out your experiment by adding chemicals or biological agents into appropriate well plate. Incubate for 6 to 24 hours. 3 . Add 10μl of the CCK-8 solution to each well of the plate. 4 . Incubate the plate for 1-4 hours in the incubator. 5 . Measure the absorbance at 450 nm using a microplate reader.

Advantages ★★★ Ready-to-use one-bottle solution No radioisotope (동위원소) or organic solvent required No toxicity to cells, CCK-8 실험후 다른 실험에 재사용 가능 No harvesting, washing or solubilization step required More sensitive than MTT, XTT, MTS or WST-1 ★★★ The major difference between CCK-8 and the MTT assay, other than MTT’s toxicity, is the enzymes involved. The CCK-8 assay involves most of the dehydrogenase(산화 환원 효소) in a cell. On the other hand, MTT only involves mitochondrial dehydrogenase (산화 환원 효소) Therefore, the MTT assay depends on mitochondrial activity, not the cell itself. Additionally, CCK-8 is far more sensitive than the MTT assay. http://www.biocompare.com/Multimedia/Video/653/Video.html

Cell proliferation assay using CCK-8 and other reagents

Product MTT Cell Proliferation Assay Cell Counting Kit-8 ATCC® Number: 30-1010K Price: $225.00 Quantity:  2500 reactions Cell Counting Kit-8 www.dojindo.com http://www.woongbee.com/0NewHome/CCK-8/090323_CCK-8.htm Tel. 031-776-3300 / Fax. 031-776-3303

LDH Cytotoxicity Detection Kit Offers a simple way to measure dead and plasma membrane- damaged cells , based on the release of lactate dehydrogenas e (LDH: a stable cytoplasmic enzyme present in most cells), due to plasm a membrane damage . Quantify cell death simply and accurately Highly sensitive cell death assay: Low cell numbers, such as 0.2 - 2 × 102 cells Streamlined method in a 96-well format, for results in under an hour Safe and convenient—no radioactive isotopes or prelabeling steps

Application Can be used in many different in vitro cell systems when damage to the plasma membrane occurs. Detection and quantification of cell mediated cytotoxicity (cytotoxic T-lymphocytes (CTL), natural killer (NK) cells, lymphokine activated killer (LAK) cells or monocytes) Determination of the cytotoxic potential of compounds in environmental and medical research. Determination of cell death in bioreactors Product Price: $378.00 Quantity:   2000 tests