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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

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    1. MTT Cell Proliferation Assay sunpingli

    2. 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.

    3. 원리

    4. 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.

    5. 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.

    6. 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

    7. 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 .

    8. 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.

    9. Key features • 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.

    10. 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.

    11. 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.

    12. 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.

    13. Cell proliferation assay using CCK-8 and other reagents

    14. Product MTT Cell Proliferation Assay • ATCC® Number: 30-1010K • Price: $225.00 • Quantity:  2500 reactions Cell Counting Kit-8 • • • Tel. 031-776-3300 / Fax. 031-776-3303

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

    16. 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