Cell Counting Kit-8 (CCK-8)
目录号 : GK10001细胞计数试剂盒-8(CCK-8)是一种即用型的单瓶溶液,可快速、可靠地测量各种化学物质对细胞存活率和毒性的敏感度。
Sample solution is provided at 25 µL, 10mM.
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细胞计数试剂盒-8(CCK-8)使用WST-8(2-(2-甲氧基-4-硝基苯基)-3-(4-硝基苯基)-5-(2,4-二磺酚基)-2H-tetrazolium,单钠盐),在存在电子载体1-Methoxy PMS的情况下,产生水溶性福尔马定染料进行方便的测定。 CCK-8溶液直接加入到细胞中,无需预先混合组分。 WST-8被细胞脱氢酶生物还原为可溶于组织培养液的橙色福尔马定产物。 产生的福尔马定量与活着的细胞数量成正比。由于CCK-8溶液非常稳定且具有很小的细胞毒性,因此可以进行更长时间(例如24至48小时)的孵育。
细胞计数试剂盒 - 8允许灵敏度高、用于增殖和毒性测定中确定存活细胞数量的显色法检测。 检测灵敏度比MTT、XTT或MTS等任何其他四唑盐都要高。
图1:Cell Counting Kit-8(CCK-8)的工作机制。
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。将细胞在CO2培养箱中于37℃培养24小时。
2.将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.在读取孔板之前,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。
常见问题
Q:如何做空白对照?
A:可以用加相应量细胞培养基和CCK-8但不加细胞的孔作为空白对照,若担心所使用的药物会干扰检测,需设置加相应量细胞培养液、药物和CCK-8但不加细胞的孔作为空白对照。
Q:只加CCK-8不加细胞的空白孔,数值上增?
A:在整个实验过程中,随着时间的增加,如果细胞培养时间较长,请注意蒸发问题。因为反应环境温度较高,会有部分液体挥发。空白组虽然没有细胞,但是随着液体的挥发,药物浓度相当于提高了,所以空白对照的数值也会有轻微的变化(但不会有明显的变化)。如果很在意空白对照的数值,可将96孔板外围一圈加培养基、水或PBS 保湿。同时,可以把96孔板置于培养箱内靠近水盘的位置以缓解蒸发。将96孔板外围两圈用PBS铺板,这样挥发先挥发周边的,可以对内部及空白对照的影响降至最低。
Q:一般多久测一次值?
A:预实验时可以在0.5、1、2和4小时后分别用酶标仪检测,然后选取吸光度范围比较适宜的一个时间点用于后续实验。
Q:加入CCK8后暂时不测OD值,可以如何处理暂时保存?能够保存多久?
A:如果需要暂时不测定OD值,可以向每孔中加入10ul 0.1M HCl溶液或者1% w/v的SDS溶液,避光保存在室温,24小时内吸光度不会发生变化。
Q:如何保存?
A:CCK-8溶液在避光、0-5℃的条件下可以保存1 年,若长期不用可在-20℃下避光保存2年。建议分装后保存于-20℃,使用时提前于4℃解冻,请避免反复冻融,否则增加背景值,干扰实验结果。
Q:是否需要更换培养基?
A:不需要,若药物影响比较小的情况可以不更换培养基,直接扣除培养基中加入药物后的空白吸收即可。
Q:待测物质有氧化性或还原性该做何处理?
A:本试剂盒的检测依赖于脱氢酶催化的反应,如果待测物质有氧化性或还原性,可在加CCK-8之前更换新鲜培养基,去掉药物的影响。
如果实验中有还原剂,请检查背景的OD值,即在不含细胞的培养基中加入药物,然后加入CCK-8试剂在一定时间内检测,和不加药物的培养基进行比较(只有CCK-8试剂),如果O.D值明显偏高,则说明有反应。
Q:如果培养基颜色或PH已变化,是否需要更换新的培养基?
A:加入CCK-8时,如果细胞培养时间较长,培养基颜色或pH值已变化,建议换用新鲜的培养基。
Q:适用细胞?
A:因为测线粒体酶,所以真核细胞,有线粒体的均可,例如单细胞,动物细胞。但有细胞壁的细胞,例如植物细胞,酵母细胞,底物进入细胞效率低,不推荐。
Q:线性不佳原因?
A:细胞不均;培养液蒸发;还原剂;气泡;孔板周围一圈不能用,除非只培养少于一天的时间;细胞数目很重要,太少信号会低。
Q:金属毒性?
A:可以检测金属毒性,最好做空白对照。
Q:适用于那些细胞?其他还有哪些细胞可用?生物被膜内的活菌 (流感嗜血杆菌和绿脓杆菌)?
A:所有哺乳动物的细胞,细菌一般没有检测CCK8的,可以染色法,或者流式,最常规的方法,有报道, 比浊法测OD600。
Q:是否可以替代Brud或EDU法检测细胞增殖?
A:虽然CCK8通过检测对生长期的细胞内的脱氢酶来反应细胞的活性,而胸腺嘧啶插入法通过核苷酸类似物的方式插入合成的DNA来检测细胞的活性,这几种方法是有一定关联性的,CCK8可替换胸腺嘧啶插入法来检测细胞增殖,并且检测更方便。
Q:空白孔数值偏高,有可能是培养基长菌吗?或者其他原因?
A:空白孔数值偏高一般认为染菌造成的,很少出现这种问题。
Q:CCK8能检测组织吗
A:不能检测组织。
Q:是否可以检测T细胞
A:可以,T细胞长的慢,需延长孵育时间。
Q:当两个细胞混合培养时(骨髓间充质细胞,另一个是悬浮T细胞),培养一段时间后,只测骨髓间充质干细胞的生长、增殖情况,请问用CCK-8能测吗?怎么做?
A:对于两种细胞的情况:
1)如果测定贴壁细胞的生长状况,那么可以把悬浮的T细胞转移掉,使用上述第二种方法测定。
2)如果T细胞对于间充质细胞的生长很关键,不能取走,那么测定的活性程度是总量,此时,需要对每一个孔的T细胞进行估算,如果每个培养条件下T细胞总量接近,或者T细胞含量很少,远远小于间充质细胞,也可以测定,使用对照孔可以去除T细胞的影响。
3)如果T细胞可以短时间内除去,可以找个灵敏度高的CCK-8,信号强,建议细胞量合适的情况下(每一个孔10000个),30分钟左右出读数结果,测 定后,可以将T细胞再加入培养孔。 这样做对细胞的生长影响不大。但是如果做动力学,那么要考虑的因素多一些。
Q:CCK-8是通过和细胞内的脱氢酶反应而反映着细胞数量,假如细胞已经死亡,而脱氢酶活性还有,那么如何计算细胞数量?
A:多数情况下,细胞死亡过程中,细胞内的成分会降解,脱氢酶会有部分释放,但是活性和活细胞比,比较低。举例说:100个细胞死亡20小时后,释放的脱氢酶活力,不足5个活细胞的脱氢酶活力,也就是说,这种情况带来的误差5%以内。而且,这种细胞死亡,对同种细胞来说,情况是一样的,一般都是在不同的孔间比较同种细胞,所以系统误差是一样的,即系统误差会彼此消减。
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 | ++ | + | + | +++ |
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