Cell Counting Kit-8 (CCK-8)
Catalog No.
K1018
用于细胞增殖和细胞毒性试验
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Separation of phosphorylated and non-phosphorylated proteins without phospho-specific antibody
Cell Counting Kit-8 (CCK-8)
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我们的产品Cell Counting Kit-8（CCK-8）相比以前的方法为细胞数测定和细胞增殖/毒性检测提供了更方便和灵敏的方法。试剂盒使用了一种高水溶性的四唑盐，WST-8，它在电子耦合试剂存在下还原产生水溶性formazan形式的染料。由脱氢酶产生的formazan的量与活细胞的数量呈直接的线性关系。CCK-8的检测灵敏度高于其他方法，如MTT，XTT，MTS或WST-1等。
CCK-8法的优势
More Sensitive than MTT, MTS, or WST-1
No Toxicity to Cell
Simpler Steps, No organic solvents required
Stable One Bottle Solution, ready-to-use
Advantages
| 100 tests | 1x1ml |
| 500 tests | 1x5ml |
| 1000 tests | 2x5ml |
| 3000 tests | 6x5ml |
| 10000 tests | 20x5ml |
| CCK-8在0-5 °C下，避光保存，储存时间超过一年。如需更长时间储存，请将其储存在-20°C。反复冻融会导致背景增加，干扰您的检测。如果频繁使用，请将试剂盒存放在0-5°C。 | |
比MTT，MTS或WST-1更敏感
对细胞无毒性
步骤更简单，无需有机溶剂
稳定的试剂，即用型
1）Q：每个孔需要接种的细胞数量是多少？
A：对于贴壁细胞，每孔至少需要1000个细胞（100 μl培养基）。对于白细胞，由于灵敏度较低，每孔至少需要2500个细胞（100 μl培养基）。推荐的96孔板每孔最大细胞数为25000。如果使用24孔或6孔板进行该检测，请计算相应的每孔的细胞数，并调整CCK-8的体积，使其为每孔总液体体积的10%。
2）Q：CCK-8对细胞的毒性大小如何？
A：CCK-8的毒性非常低，在CCK-8测定完成后，相同的细胞可用于其他细胞增殖测定，例如结晶紫测定，中性红测定或DNA荧光测定。（除非细胞极为稀少，不推荐把CCK-8测定过的细胞用于其它实验。）
3）Q：怎么设置空白对照？
A：在不含有细胞的培养基中加入CCK-8，酶标仪检测450 nm的吸光度；若做药物刺激实验，可在不含细胞、含药物的培养基中加入CCK-8。酶标仪检测450 nm的吸光度作为空白对照。
4）Q：CCK-8能否用于细胞染色？
A：CCK-8不能用于细胞染色，CCK-8检测原理是高水溶性的四唑盐WST-8在电子耦合试剂存在下还原产生水溶性formazan形式的染料，WST-8及formazan是高度水溶性的，不会进入细胞内对细胞进行染色。
5）Q：没有450 nm的滤光片，能否使用其他滤光片？
A：如果您没有450 nm滤光片。您也可以使用吸光度在430 nm和490 nm之间的滤光片, 450 nm滤光片具有最佳灵敏度。
6）Q：哪些物质会干扰CCK-8的测定？
A：WST-8可能与还原剂反应生成WST-8 formazan，如果使用还原剂（例如一些抗氧化剂）会增加OD值；若存在氧化性物质则会抑制CCK-8测定反应，减小OD值；此外，培养基中的酚红不会影响实验结果，酚红的吸光度可以在计算时，通过扣除空白孔中本底的吸光度而消去。
7）Q：在做药物刺激实验时，药物对测定是否有影响？
A：如果药物有还原性，则会与CCK-8发生反应，影响吸光度；药物中金属离子的存在可能会影响CCK-8的灵敏度。终浓度为1 mM 的氯化亚铅、氯化铁、硫酸铜会抑制5% 、15% 、90%的显色反应，使灵敏度降低。如果终浓度是10 mM 的话，将会100%抑制。
8）Q：如果吸光度值太低，可以采取什么办法？
A：适当增加细胞数量或延长加入CCK-8溶液后的孵育时间。
9）Q：如果吸光度值太高，可以采取什么办法？
A：适当减少细胞数量（建议正式实验前优化细胞数量与荧光值之间的关系）或缩短加入CCK-8溶液后的孵育时间。