Cell Counting Kit-8，CCK-8细胞增殖检

1.细胞数量过低，建议增加细胞待测数量；
2.染色难度大，建议增加CCK-8孵育时间

◆数据重复性差？
1.孔板最外圈试剂容易挥发，建议最外一圈的孔只加培养基，不作为测定孔用；
2.CCK-8沾壁，建议加入CCK-8后，轻轻敲击培养板以帮助混匀；
3.细胞数量过多或过少，建议预先在1000-100000个/孔范围内摸索条件；
4.孔内气泡干扰酶标仪检测，检测前需确保每个孔内无气泡

◆在做细胞的加药实验时,药物对CCK-8测定是否有影响?
1.注意如果药物具有还原性(如维生素C),会和CCK-8发生显色反应,增加吸光度；
2.药物中的金属对CCK-8显色有影响:当终浓度为1mM的氯化亚铅、氧化铁、硫酸铜会抑制5%,15%, 90%的显色反应,使灵敏度降级。如果终浓度是10mM的话，将会100%抑制。解决办法:首先要确认药物是否有吸收,在含有药物的培养基中加入CCK-8,测定450nm的吸光度,如果它的吸光度比不含药物的培养基(加CCK-8)的吸光度高,则证明药物有影响,可在加CCK-8之前更换培养基,去掉药物的影响；
3.由于培养基中可能含有氧化还原反应的物质,在正式实验之前,建议使用一个孔作一下检测,有必要先确认培养基和CCK-8是否反应，一般正常的OD值应该在0.4以下

◆可否采用CCK-8法进行药物的IC50检测？
可以,将细胞培养后按照不同浓度的药物处理一定时间后进行CCK-8检测,根据吸光度绘制曲线,计算该药物抑制细胞数量一半时的浓度(IC50)

◆CCK-8对于不同的细胞,灵敏度是否一样？
不一样,悬浮细胞较贴壁细胞难染色。对于贴壁细胞,一般加入CCK-8培养1-4小时吸光度已经很高,但对于悬浮细胞则可能吸光度较低,可以通过延长CCK-8的加入时间或增加细胞数量来解决

◆若是暂不检测OD值，该如何处理?
若暂时不测定 OD 值，可以向每孔中加入 10μL 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

◆用CCK-8测细胞活力时每次都要做标准曲线吗？
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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