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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Cell Host & Microbe 32.1 (2024): 48-62. PMID: 38056458 IF: 30.3013 -
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Fe3O4@IR820@CpG induces immunogenic cell death and promotes DC maturation in vitro. A,B) Viability of MC38 cells after incubation with A) different concentrations of Fe3O4@IR820@CpG without laser irradiation or B) different formulations with 808 nm laser irradiation (1 W cm−2) for 5 min, as detected by CCK-8 kit (n = 4).
Bicinchoninic Acid Assay (BCA) protein assay kit,cell counting kit-8 (CCK-8), and erythrocyte membrane lysates were purchased from GlpBio (Montclair, USA).
Advanced Materials, 2023: 2307193. PMID: 37951210 IF: 29.4006 -
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H) The viability ofMC38 cells treated with PAD@MS for 48 h in the presence or absence of 10 μM Ferr-1, 20 μM Nec-1, 10 μM z-VAD-FMK, and 1 mM NAC.
Cell viability was detected by cell counting kit-8 (CCK-8) assay (GlpBio, USA) according to the manufacturer’s instruction.
Adv Funct Mater, 2023: 2214998. IF: 19.9246 -
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After transfection with si-1 and si-2, performed CCK-8 assays were performed to evaluate the cellular growth curves and the results revealed that repressing the expression of PCAT6 remarkably suppressed the cell proliferation of HeLa and SiHa cells.
Each well was added with 10 μl CCK-8 reagent (GLPBIO, Montclair, CA, USA) and the plates were continued to be cultured for 1-2 h. The cell viability was determined by measuring the optical density (OD) absorbance at the wavelength of 450 nm.
Eur Rev Med Pharmacol Sci.2019 Mar;23(5):1947-1956 -
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Blocking bFGF leads to decrease in cell proliferation and clone formation of cisplatin (DDP) selected A549 cells. Growth curves showed that knocking down bFGF expression resulted in reduction in cell proliferation.
The cells were incubated for 24 h in incubator, and then,10μl CCK-8 (GLPBIO) was added in every well and cultivated at room temperature for 4 h. Absorbance was measured at 450 nm. The experiment repeated three times successive 7 days.
J Pharm Pharmacol.2019 Sep;71(9):1412-1420.