CCK-8，您细胞活力检测的专家

——灵敏、简便、低毒

关键词：细胞增殖；细胞毒性；CCK-8；MTT

产品背景

细胞功能实验中，对于细胞增殖、毒性和活力的检测是至关重要的。目前常用的细胞增殖与活性检测试剂通常是通过基于线粒体内的脱氢还原酶的还原能力来进行检测的，如MTT法，XTT法，WST-1法，CCK 法等。

翊圣生物推出一款基于WST-8的细胞活性检测试剂盒—Cell Counting Kit简称CCK试剂盒，相较于MTT等其细胞毒性更低，检测更灵敏，操作更简便。

实验原理

WST-8（2-(2-甲氧基-4-硝苯基)-3-(4-硝苯基)-5-(2,4-二磺基苯)-2H-四唑单钠盐）在电子耦合载体1-Methoxy PMS存在的情况下，可以被线粒体内的脱氢酶还原生成高度水溶性的橙黄色甲臜产物（formazan），颜色的深浅与细胞的增殖成正比，与细胞毒性成反比。使用酶标仪在450nm波长处测定OD值，间接反映活细胞数量。

表1 CCK法与其他细胞增殖/毒性检测方法的优势比较

产品特点

操作简单：产品本身是液体，即开即用；甲臜产物是水溶性的，不需要用有机溶剂溶解。

灵敏度高：线性范围广，灵敏度高，重复性好。

细胞毒性低：细胞毒性非常低，对后续实验没有影响。

稳定性好：培养液中酚红和血清的存在不会对检测造成影响。

应用性广：可用于大规模、高通量样品检测。

适用范围

细胞增殖测定、细胞毒性、药物筛选、肿瘤药敏试验、生物因子的活性检测等

产品数据

同类产品性能比较：

细胞类型：293T人胚肾细胞；培养基：10% FBS 高糖DMEM培养基

孵育时间：3h；细胞数目：1000

引用已发表文献数据

FAQ

Q1：CCK的保存和稳定性如何？

答：CCK稳定性高，避光条件-20℃有效期2年，4℃有效期1年。经常使用建议4℃保存，避免反复冻融。CCK正常颜色为粉红色，若颜色发生明显偏差，质量可能有发生变化。

Q2：CCK的细胞毒性如何？

答：CCK毒性非常低，因此CCK检测完后相同的细胞还能用于其它细胞增殖检测如结晶紫染色法，中性红染色法或DNA荧光染色法。

Q3：CCK会对活细胞进行染色吗？

答：不会，首先WST-8不会进入细胞染色，整个显色反应都是在培养体系中进行的。另外WST和WST-8甲臜都属于高度水溶性组分，反应结束更换新的培养液，即可清除外源组分。

Q4：做细胞活性检测时，每孔应该接种多少个细胞？

答：当使用标准96孔板时，贴壁细胞的最小接种量至少为1,000个/孔(100 μL培养基)。检测白细胞时的灵敏度相对较低，因此推荐接种量不低于2,500个/孔(100 μL培养基)。

Q5：若是使用6孔或者24孔板进行试验，CCK试剂使用量怎么样呢？

答：如果要使用6孔板或24孔板实验，请先计算每孔相应的接种量，并按照每孔培养基总体积的10%加入CCK溶液。

Q6：培养基的本底颜色如酚红会不会影响细胞活性的检测呢？

答：不会的，培养基中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值而消去，因此不会对检测造成影响。

Q7：能否用384孔板进行细胞增殖与活性检测的实验呢？

答：可以，CCK-8产品的使用量为每孔培养基总体积的10%。建议先将CCK-8产品稀释一倍，然后加入培养基总体积的20%即可，这样可减少误差。

Q8：只可以在450 nm波长处测OD值吗？

答：可以在450 nm波长处测OD值，但是若无此波长的滤光片，可选择吸光度在430-490 nm之间的滤光片，但是450 nm滤光片的检测灵敏度最高。

Q9：若是暂不检测OD值，该如何处理？

答：若暂时不测定OD值，可以向每孔中加入10 μL 0.1 M的HCL溶液或者1% w/v SDS溶液，并遮盖培养板避光保存在室温条件下。24小时内测定，吸光度不会发生变化

Q10：在加入CCK-8时可否不更换培养基？

答：一般情况下可以不更换培养基。但是如果培养基里有氧化还原性物质的话，有可能会产生误差，必须更换其它培养基。

Q11：在实验中OD值太高，怎么解决？

答：可以缩短加入CCK-8后的培养时间。例如：可以把加入CCK-8试剂后的培养时间由2小时缩短为1小时。此外，可适当减少细胞的数量。

Q12：如果OD值太低，怎么解决？

答：可以采取2个办法：1、适当增加细胞数量  2、延长加入CCK-8试剂后的反应时间。

Q13：应该每次做标准曲线吗？

答：建议每次做。虽然细胞是一样的，但是细胞的状态不一定一样。对于状态不一样的细胞，建议每次做标准曲线。

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