Cell Counting Kit-8 (CCK-8) 目录号 GK10001 |
Sample solution is provided at 25 µL, 10mM.
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Related Biological Data

Related Biological Data

Related Biological Data

细胞数量确定
1.将细胞悬浮液(100μL/孔)接种在96洞孔板中。将板在潮湿的培养箱中预孵育(例如,在37℃,5%CO2下)。
2.向板的每个孔中加入10μLCCK8溶液。注意不要在孔洞中引入气泡,因为它们会干扰O.D.读数。
3.将培养板在培养箱中孵育1-4小时。
4.使用酶标仪测量450 nm处的吸光度。
细胞增殖和细胞毒性测定
1.将种子细胞以103-104个细胞/孔的密度在96孔板中在100μL培养基中培养,含有或不含有待测化合物。将细胞在CO2培养箱中于37℃培养24小时。
2.将10μL不同浓度的待测物质加入板中。
3.将培养板在培养箱中孵育适当的时间(例如,6,12,24或48小时)。
4.使用重复移液器向板的每个孔中加入10μLCCK8溶液。注意不要在孔洞中引入气泡,因为它们会干扰O.D.读数。
5.将培养板在培养箱中孵育1-4小时。
6.在读取印版之前,重要的是在轨道振动器上轻轻混合1分钟,以确保颜色均匀分布。
7.使用酶标仪测量450 nm处的吸光度。
数据分析
统计分析有几种方法,您可以选择使用O.D.值或细胞数量,我们提供其中一种方法。
细胞存活率(%)= [(As-Ab)/(Ac-Ab)]×100
抑制率(%)= [(Ac-As)/(Ac-Ab)]×100
As =实验孔吸光度(含有细胞,培养基,CCK-8和待测化合物的孔的吸光度)。
Ab =空白孔吸光度(含有培养基和CCK-8的孔的吸光度)。
Ac =对照孔吸光度(含有细胞,培养基和CCK-8的孔的吸光度)。
制作标准曲线
1. 细胞计数板计数细胞悬液中的细胞数。
2. 使用培养基,等比稀释细胞悬液为一个浓度梯度,通常需要5-7个浓度梯度,每组几个复孔。然后接种细胞。(注意每孔的细胞数量。如果您将细胞悬液稀释在管中,在加入培养板的孔之前,请小心再次混匀细胞。每孔中细胞悬液的体积应该是一致的。)
3. 培养直至细胞贴壁(通常2-4小时),然后每100 μl培养基加入10 μl CCK-8。继续孵育1-4小时,用酶标仪测量450nm处的吸光度。制作出一条以细胞数为X轴坐标,O.D.值为Y轴坐标的标准曲线。
可以基于该曲线确定待测样品的细胞数。使用此标准曲线的先决条件是培养检测条件相同。
注意事项
1. 确保药物和CCK8均匀分布在培养基中。
2. 细胞增殖越多, 颜色越深; 细胞毒性越强,颜色越浅。
3. 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%。
4. 因为CCK8测定是基于活细胞中的脱氢酶活性,影响脱氢酶活性的条件或化学物质可能导致实际活细胞数与使用CCK-8测定活细胞数之间有差异。
5. 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)会干扰检测。如果待检测体系中存在较多的还原剂,需设法去除。
6. 孵育2小时后,背景O.D.值一般为0.1-0.2单位。
7. 注意不要在孔中引入气泡,因为它们会干扰O.D.值。
8. 如果您想对CCK8溶液进行灭菌,请使用0.2 μm的膜过滤溶液。
9. 孵育时间因孔中细胞的类型和数量而异。通常,白细胞着色较弱,因此可能需要较长的孵育时间(长达4小时)或大量细胞(~105细胞/孔)。
10. 如果细胞悬液中存在高浊度, 测量并减去样品在600nm或更高波长的O.D值。
11. CCK8不能用于细胞染色。
12. 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,因此不会对检测造成影响。
13. CCK8的毒性非常低,在CCK8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。)
14. 该试剂盒可用于大肠杆菌,但不能用于酵母细胞。
15. 在读取平板之前,您可以在摇床上轻柔混匀。
16. 我们建议将细胞接种在靠近培养板中央的孔中,最外围一圈孔中的培养基容易蒸发,可以用PBS,水或培养基填充这些孔。
17. 如果您没有450nm滤光片。您也可以使用吸光度在430和490nm之间的滤光片, 450nm滤光片具有最佳灵敏度。
18. 测量450nm处的吸光度,如果您需要进行双波长测定,作为参考波长可以测定650nm处的吸光度。
19. 药物中金属离子的存在可能会影响CCK8的灵敏度。终浓度为1mM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10mM的话,将会100%抑制。
应用&特点 |
1) 细胞增殖测定 - GlpBio细胞计数试剂盒-8(CCK-8)是水溶性的,在培养中是稳定的,并且是无毒的。 2) 细胞活力测定 - 代谢活性和染料产生与改变的活力成比例地变化。 3) 细胞因子测定 - 测量细胞因子诱导的增殖。如果需要,可以在研究结束时回收细胞并扩增。 4) 细胞毒性测定 - 来自细胞毒性化学物质的细胞死亡对颜色发展没有影响,只有活细胞将试剂转化为比色指示剂。试剂本身具有可忽略的毒性,并且通常对细胞是安全的。 |
运输方式 | 蓝冰运输。 |
储存条件 | 储存在4°C避光,可稳定长达12个月。储存在-20°C避光,可稳定长达2年。 |
用途 | 仅供研究使用!不能用于人体。 |
CCK-8 | MTT | MTS | SRB |
Solubility | Water soluble | Indissolvable | Water soluble | Indissolvable |
Detection Wavelength | 450nm | 490nm | 450nm | 510nm |
Character | Liquid | Solid | Liquid | Liquid |
Usage | No need to prepare | Prepare the solutions | Use it right after it was ready | Prepared beforehand |
Need to redissolve or not | NO | Yes,by DMSO | No | Yes,by Tris-base solution |
Convenience | +++ | ++ | +++ | + |
Detection speed | +++ | + | ++ | + |
Repeatability | +++ | + | ++ | ++ |
Stability | ++ | + | + | +++ |
细胞计数试剂盒-8(CCK-8)允许使用WST-8(2-(2-甲氧基-4-硝基苯基)-3-(4-硝基苯基)-5-(2,4-二磺基苯基)-2H-)进行方便的分析四唑盐,一钠盐),其在电子载体1-甲氧基PMS存在下生物还原时产生水溶性甲dye染料。将CCK-8溶液直接添加到细胞中,不需要预先混合组分。 WST-8被细胞脱氢酶生物还原为可溶于组织培养基的橙色甲臜产物。产生的甲的量与活细胞的数量成正比。由于CCK-8溶液非常稳定并且几乎没有细胞毒性,因此可以进行更长的孵育,例如24至48小时。
细胞计数试剂盒-8允许灵敏的比色测定法测定增殖和细胞毒性测定中活细胞的数量。检测灵敏度高于任何其他四唑盐,如MTT,XTT或MTS。
图1: Cell Counting Kit-8 (CCK-8)的工作机制.