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MTT细胞增殖及细胞毒性检测试剂盒可用于细胞增殖和细胞毒性的检测。MTT可被线粒体内的一些脱氢酶还原生成结晶状的深紫色产物甲瓒。在特定溶剂存在的情况下，可被完全溶解。然后通过酶标仪可以测定570nm波长附近的吸光度。细胞增殖越多越快，则吸光度越高；细胞毒性越大，则吸光度越低。
使用方法
1. MTT溶液的配制：用5mL MTT Solvent溶解25mg MTT，配制成5mg/mL的MTT溶液。在无菌环境下，用0.22μm过滤后使用，或直接-20℃避光保存，也可以根据需要适当分装后-20℃避光保存。
2. 取生长状态良好的细胞，调整细胞密度，按每孔100 μL细胞悬液接种于96孔板中，同时设空白孔（不加细胞但是加入同体积的培养基）。
注：通常细胞增殖实验每孔加入100 μL约含2000个细胞，细胞毒性实验每孔加入100 μL约含5000个细胞（具体每孔所用的细胞的数目，需根据细胞的大小，细胞增殖速度的快慢等决定）。
3. 根据实验设计对细胞进行培养和处理。
4. 每孔加入10 μL MTT溶液，在细胞培养箱内继续孵育4小时。
5. 对于贴壁细胞，直接吸掉旧的培养液，避免枪尖刮破单细胞层。对于悬浮细胞，离心收集细胞，去除培养液。
6. 每孔加入150 μL Formazan Solvent，适当混匀，在细胞培养箱内再继续孵育。直至在普通光学显微镜下观察发现formazan全部溶解。 通常37℃孵育15-30分钟左右，紫色结晶会全部溶解。如果紫色结晶较小较少，溶解的时间会短一些。如果紫色结晶较大较多，溶解的时间会长一些，此时为加速溶解可以适当振摇数次。
7. 镜下观察，甲臜充分溶解后用酶标仪测定在570 nm处的吸光度。
注意事项
1. 本产品仅供科研使用。
2. 为了您的安全与健康，请穿戴实验室工作服和一次性手套进行操作，并遵守实验室试剂操作规程。
3. 由于使用96孔板进行检测，如果细胞培养时间较长，一定要注意蒸发的问题。一方面，由于96孔板周围一圈最容易蒸发，可以采取弃用周围一圈的办法，改加PBS，水或培养液；另一方面，可以把96孔板置于靠近培养箱内水源的地方，以缓解蒸发。
4. MTT Solvent 在低温情况下会凝固，使用前请室温放置或20-25ºC水浴温育片刻至全部融解后使用。
5. MTT配制成溶液后为黄色，需避光保存，长时间光照会导致失效。当颜色变为灰绿色时，请勿使用。MTT溶液在4ºC、冰 浴等较低 温度情况下会凝固，可以20-25ºC水浴温育片刻至全部融解后使用。
6. 细胞接种时注意混匀，避免细胞沉降导致每孔中细胞接种数量不一致。
7. Formazan Solvent 结冻或产生沉淀时可以 37°C 水浴孵育以促进溶解，并且必须在全部溶解并 混匀后使用。
8. 细菌、支原体或其他微生物可破坏 MTT 四唑环，污染的细胞不能使用该方法进行检测。
The MTT cell proliferation and cytotoxicity detection kit can be used for the detection of cell proliferation and cytotoxicity. MTT can be reduced by some dehydrogenases in the mitochondria to form a crystalline, dark purple product called methylene blue. In the presence of a specific solvent, it can be completely dissolved. Then, the absorbance at 570nm wavelength can be measured using an enzyme reader. The more and faster the cell proliferation, the higher the absorbance; the greater the cytotoxicity, the lower the absorbance.
Usage method
1. Preparation of MTT solution: Dissolve 25 mg of MTT in 5 mL of MTT solvent to prepare a 5 mg/mL MTT solution. Use it in a sterile environment after filtration through a 0.22 μm filter, or store it at -20°C in the dark. It can also be appropriately aliquoted and stored at -20°C in the dark as needed.
2. Take cells in a good growth state, adjust the cell density, and inoculate 100 μL of cell suspension into each well of a 96-well plate. At the same time, set a blank well (without cells but with the same volume of culture medium).
Note: Usually, in a cell proliferation experiment, 100 μL of approximately 2000 cells is added to each well, and in a cell toxicity experiment, 100 μL of approximately 5000 cells is added (the exact number of cells used in each well depends on the size of the cells and the speed of cell proliferation, etc.).
3. Cultivate and treat the cells according to the experimental design.
4. Add 10 μL of MTT solution to each well, and incubate in the cell incubator for another 4 hours.
5. For adherent cells, simply remove the old culture medium directly to avoid the pipette tip from scratching the single-cell layer. For suspended cells, centrifuge to collect the cells and remove the culture medium.
6. Add 150 μL of Formazan Solvent, mix appropriately, and continue incubating in the cell incubator. Until all the formazan is completely dissolved under a normal optical microscope. Usually, incubate at 37°C for about 3-4 hours, and the purple crystals will all dissolve. If the purple crystals are smaller and fewer, the dissolution time will be shorter. If the purple crystals are larger and more numerous, the dissolution time will be longer. At this time, to accelerate the dissolution, you can shake several times appropriately.
7. Observe under a microscope, and measure the absorbance at 570 nm using an enzyme detector after the formazan is fully dissolved.
Matters needing attention
1. This product is for scientific research use only.
2. For your safety and health, please wear laboratory work clothes and disposable gloves during operation, and follow the laboratory reagent operation procedures.
3. Since the detection is carried out using 96-well plates, if the cell culture time is long, be sure to pay attention to the evaporation problem. On the one hand, since the outer circle of the 96-well plate is the most prone to evaporation, you can adopt the method of discarding the outer circle and adding PBS, water or culture medium; on the other hand, you can place the 96-well plate near the water source inside the incubator to alleviate evaporation.
4. The MTT solvent will solidify at low temperatures. Before use, please let it stand at room temperature or incubate in a water bath at 20-25°C for a moment until it is completely dissolved before using.
5. After preparing the MTT solution, it is yellow and needs to be stored in the dark. Long-term exposure to light will cause it to lose its effectiveness. When the color turns grayish green, do not use it. The MTT solution will solidify at 4°C, in an ice bath, etc., and can be incubated in a water bath at 20-25°C until it is completely dissolved before using.
6. When inoculating cells, pay attention to mixing well to avoid cell sedimentation, which may cause inconsistent cell inoculation amounts in each well.
7. When the Formazan Solvent is frozen or has precipitation, it can be incubated in a 37°C water bath to promote dissolution, and must be used after all is dissolved and mixed.
8. Bacteria, mycoplasma or other microorganisms can damage the MTT tetrazolium ring, and contaminated cells cannot be used for this method of detection.
C1375225 | Component | 500T | 2500T | Storage |
C1375225A | MTT | 25 mg | 125 mg | 2-8℃. Store in the dark. |
C1375225B | MTT Solvent | 5 mL | 25 mL | 2-8℃. |
C1375225C | Formazan Solvent | 80 mL | 5×80 mL | 2-8℃. Store in the dark. |
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| 批号(Lot Number) | 证书类型 | 货号 |
|---|---|---|
 ZJ26F0838721 |
分析证书 | C1375225 |
| ZJ26F0131755 |
分析证书 | C1375225 |
| ZJ26F0131754 |
分析证书 | C1375225 |
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