MTT assay (WST-8 assay)

본 내용은 " 세포생물학실험 만점(A+) 실험 보고서(레포트) 04. MTT assay (WST-8 assay) " 의 원문 자료에서 일부 인용된 것입니다. 2024.07.09

문서 내 토픽

• 1. Tetrazolium

  Tetrazolium은 dehydrogenase 등의 효소에 의해 쉽게 환원되어 유색의 formazan을 형성한다. 이러한 원리로 가장 널리 사용되는 MTT tetrazolium은 노란색 수용성 기질로, 미토콘드리아에 있는 reductase에 의해 환원되어 청자색을 띄는 비수용성의 MTT formazan 이라고 불리는 crystal을 형성한다. 따라서 세포사멸에 의해 미토콘드리아가 제 기능을 하지 못하게 되면 MTT를 처리해도 formazan을 형성하지 못하게 된다.

• 2. MTT assay

  MTT assay는 약리학, 독성학 등의 다양한 실험실에서 특정 물질이, 특정한 생물학적, 생화학적 기능을 50% 억제하는데 필요한 양을 나타내는 IC50을 측정하기 위한 용도로 널리 사용되고 있다.

• 3. WST-8 assay

  WST-8는 테트라졸리움(Tetrazolium) 계열의 구성물질로, 살아있는 세포에서 미토콘드리아 탈수소효소(dehydrogenase)에 의해 환원되어서 오렌지 계열의 포르마잔(formazan)을 형성한다. 이때, 생성된 포르마잔은 수용성이고, 쉽게 용해되는 특성을 지니고 있다. 즉, 미토콘드리아의 활성 측정을 통해서, 실험에 사용한 화합물이 세포에 얼마나 독성을 나타내는지 확인하는 것이다.

• 4. Docetaxel

  Docetaxel은 유방암, 난소암 등 다양한 암을 치료하기 위해 사용되는 항암제로, 탁센 계열의 약물이다. 도세탁셀은 미세소관을 형성하는 튜블린과 결합하여, 미세소관의 조립을 촉진하고, 해체는 억제하는 역할을 수행하여 세포분열이 일어나지 못하게 막는 방법으로 암세포의 증식을 억제한다.

• 5. ELISA

  ELISA는 'enzyme-linked immunosorbent assay'의 약자로 항원-항체의 특이적 결합반응과 효소를 이용하는 발색반응으로, 물질을 정성, 정량 분석하는 방법이다. ELISA는 주로 96-well flat bottom plate에서 실시되며, 각 well에서 흡광도를 측정하는 방법을 통해서 분석물의 양을 검사할 수 있다.

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• 1. Tetrazolium

  Tetrazolium is a widely used class of chemical compounds in various biological and biochemical applications. These compounds are commonly used as redox indicators, where they undergo reduction reactions to produce colored formazan products. The tetrazolium assay is a popular method for measuring cell viability, proliferation, and cytotoxicity in cell culture experiments. The assay relies on the ability of living cells to reduce the tetrazolium salt, such as MTT or WST-8, into a colored formazan product. The amount of formazan produced is directly proportional to the number of viable cells, allowing researchers to quantify cell growth, proliferation, and the effects of various treatments on cell health. Tetrazolium-based assays are versatile, sensitive, and relatively simple to perform, making them valuable tools in fields like cell biology, drug discovery, and toxicology. However, it is important to understand the limitations and potential artifacts associated with these assays, as the reduction of tetrazolium salts can be influenced by various factors beyond just cell viability. Careful experimental design and appropriate controls are necessary to ensure the reliability and interpretation of tetrazolium-based assay results.

• 2. MTT assay

  The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay is a widely used colorimetric method for measuring cell viability and proliferation. It is a type of tetrazolium-based assay that relies on the reduction of the yellow MTT salt by metabolically active cells to produce a purple formazan product. The amount of formazan formed is directly proportional to the number of viable cells, allowing researchers to quantify the effects of various treatments, drugs, or environmental conditions on cell health and proliferation. The MTT assay is relatively simple, sensitive, and can be easily adapted to high-throughput screening applications. It has been extensively used in various fields, including cell biology, pharmacology, toxicology, and drug discovery. However, it is important to note that the MTT assay has some limitations, such as potential interference from certain compounds, the need for solubilization of the formazan product, and the potential for false-positive or false-negative results under certain conditions. Careful experimental design, appropriate controls, and an understanding of the assay's limitations are crucial for the accurate interpretation of MTT assay results.

• 3. WST-8 assay

  The WST-8 (2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt) assay is another type of tetrazolium-based cell viability and proliferation assay. Similar to the MTT assay, the WST-8 assay relies on the reduction of the WST-8 tetrazolium salt by metabolically active cells to produce a water-soluble formazan dye. The amount of formazan produced is directly proportional to the number of viable cells, allowing researchers to quantify the effects of various treatments, drugs, or environmental conditions on cell health and proliferation. The WST-8 assay has several advantages over the MTT assay, including improved water solubility of the formazan product, reduced interference from certain compounds, and a more stable and sensitive signal. Additionally, the WST-8 assay is generally considered less toxic to cells compared to the MTT assay. These features make the WST-8 assay a popular choice for cell-based assays, particularly in high-throughput screening applications and studies involving sensitive cell lines. However, as with any tetrazolium-based assay, it is essential to understand the potential limitations and sources of interference to ensure accurate interpretation of the results.

• 4. Docetaxel

  Docetaxel is a widely used chemotherapeutic agent that belongs to the taxane class of drugs. It is primarily used in the treatment of various types of cancer, including breast, lung, prostate, and ovarian cancer. Docetaxel works by disrupting the normal function of microtubules, which are essential components of the cell's cytoskeleton. By binding to and stabilizing microtubules, docetaxel prevents their disassembly, leading to cell cycle arrest and ultimately cell death. The use of docetaxel has significantly improved the prognosis and survival rates for many cancer patients. However, like other chemotherapeutic agents, docetaxel can also have significant side effects, such as neutropenia, febrile neutropenia, peripheral neuropathy, and fluid retention. Careful patient selection, dose optimization, and management of side effects are crucial for the effective and safe use of docetaxel in cancer treatment. Additionally, ongoing research is exploring combination therapies, novel drug delivery systems, and the development of next-generation taxane derivatives to improve the efficacy and tolerability of docetaxel-based cancer treatments.

• 5. ELISA

  ELISA (Enzyme-Linked Immunosorbent Assay) is a widely used analytical technique in various fields, including immunology, diagnostics, and biomedical research. It is a sensitive and specific method for the detection and quantification of a wide range of analytes, such as proteins, hormones, antibodies, and other biomolecules. The ELISA assay relies on the specific interaction between an antigen and an antibody, which is then detected and quantified through an enzymatic reaction that produces a measurable signal. ELISA has several advantages, including high sensitivity, specificity, and the ability to analyze multiple samples simultaneously. It is commonly used for the diagnosis of infectious diseases, the detection of biomarkers, the evaluation of immune responses, and the quantification of therapeutic drug levels. ELISA has also been instrumental in the development of various diagnostic kits and the monitoring of disease progression and treatment efficacy. However, it is important to consider the potential limitations of ELISA, such as the possibility of cross-reactivity, matrix effects, and the need for careful optimization and validation of the assay. Proper experimental design, the use of appropriate controls, and the interpretation of ELISA results in the context of the specific application are crucial for ensuring the reliability and accuracy of the data.