

Cell Counting Kit-8 (CCK-8) 作为MTT法的替代方法,可对细胞增殖和细胞毒性进行快速、高灵敏度、无放射性的比色检测,广泛应用于抗肿瘤药物筛选、生物活性因子检测、细胞增殖或毒性测定、肿瘤药敏等试验。
CCK-8试剂无需预配即可直接加入细胞样品中,试剂中含有WST-8 [2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium,monosodium salt],WST-8在电子耦合试剂(1-Methoxy PMS)的作用下被线粒体内的脱氢酶还原生成高度水溶性的橙黄色Formazan,产物数量与活细胞数量呈线性关系,通过测定450 nm处吸光值的变化即可反映活细胞的数量。
①CCK-8的培养时间一般为1-4小时,需要摸索接种细胞的数量和加入CCK-8试剂后的培养时间,CCK-8的最佳反应时间以具体显色最佳时间为准;
②孵育时间因细胞类型和数量而异;白细胞着色较弱,可能需要较长的孵育时间或大量细胞;
③当使用标准96孔板时,贴壁细胞的最小接种量为1000个细胞/孔(100 μL培养基);检测白细胞时的灵敏度相对较低,推荐接种量应不低于2500个细胞/孔(100 μL培养基);如果要使用24孔板或6孔板,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-8溶液;
④如果没有450 nm滤光片,可以使用430-490 nm之间的滤光片,但450 nm检测灵敏度最高。如果您需要进行双波长测定,650 nm处的吸光度可作为参考波长进行测定;
⑤培养基中的酚红不会影响实验结果,酚红的吸光度可以通过扣除空白孔中本底的吸光度而消去;
⑥本产品可以检测E.coli,但不能检测酵母细胞;
⑦如果您需要对CCK-8溶液进行无菌处理,请使用0.2 μm滤膜过滤除菌;
⑧使用96孔板进行检测时,96孔板外圈孔位易蒸发,如果细胞培养时间较长,可以选择弃用外圈孔位,或改加等量的PBS、水或培养液作为填充液。
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