计算溶液所需的质量、体积或浓度。
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，适用于对基线干扰要求严格的色谱和分析工作流程。
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文献证明
在色谱分析、有机合成和交叉偶联反应领域已被 0 篇同行评审文献引用。
概述
细胞活力和细胞毒性测定通常用于药物筛选和化合物细胞毒性测试。CCK-8试剂盒使用高度水溶性的四氮盐 (称为WST-8) 来产生适用于细胞增殖和细胞毒性测定的水溶性WST-8甲攒。与MTT不同，WST-8和WST-8甲攒在细胞培养基中没有细胞毒性，因此可以使用同一检测板进行多个下游实验。 CCK-8法是一种便捷的比色测定法，用于测定细胞活力，无需增溶过程，只需最少的步骤即可提供结果。CCK-8法可用于96孔微孔板测定以及高通量筛选384孔微孔板测定。
优势：
目前市售液体型CCK-8试剂盒普遍存在贮存条件苛刻(-4C或-20)，在不同pH范围下使用不稳定，易变质(变色或产生沉淀)等缺陷。固体即溶型CCK-8试剂盒采用全新配方，瑞士工艺，克服了液体型CCK-8试剂盒的这些缺点，可以在室温下长期保存(>3年)，即开即用，在宽pH范围内稳定，实验结果更可靠。与液体型CCK-8试剂盒相比，固体即溶型CCK-8试剂盒的灵敏度更高，生物响应时间缩短一半。
应用范围：
可用于药物筛选、细胞增殖测定、细胞毒性测定、肿瘤药敏试验以及生物因子的活性检测。
操作说明：
本试剂盒可以用于药物筛选、细胞增殖测定、细胞毒性测定、肿瘤药敏试验以及生物因子的活性检测。
1.沿包装袋缺口处小心缓慢撕开；
2.将袋内粉末全部倒入装有10mL超纯水的干净容器中,持 续摇晃1分钟,当固体完全溶解后即可使用；
3.未使用完的试剂务必在4℃以下低温保存。
检测所需设备：
带有 450 - 490 nm 过滤器的酶标仪96孔板；
二氧化碳培养箱；
96 孔板，用于细胞检测的灭菌透明板；
多通道移液器（8 或 12 通道：10-100 µl）；
血细胞计数器或细胞计数器。
细胞活性检测：
1. 在96孔板中接种细胞悬液 (100 μl/孔), 将培养板放在二氧化碳培养箱中预培养24小时 (37℃, 5% CO2)；
2. 向每孔加入10 μl CCK-8溶液 (注意不要在孔中生成气泡，会影响OD值的读数)；
3. 将培养板在培养箱内孵育1-4小时；
4. 用酶标仪测定在450 nm处的吸光度；
5. 若暂时不测定OD值，可以向每孔中加入10 μl 0.1M的HCI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。
细胞增殖-毒性检测：
1. 在96孔板中接种细胞悬液 (100μl/孔), 将培养板放在培养箱预培养24小时 (37℃, 5% CO2)；
2. 向培养板加入10ul不同浓度的待测物质；
3. 将培养板在培养箱孵育一段适当的时间 (例如: 6、12、24或48小时);
4. 向每孔加入10 μl CCK-8溶液 (注意不要在孔中生成气泡，它们会影响OD值的读数);
5. 将培养板在培养箱内孵育1-4小时;
6. 用酶标仪测定在450nm处的吸光度;
7. 若暂时不测定OD值，可以向每孔中加入10 μl 0.1M的HCI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。
细胞存活率/抑制率计算方法：
细胞存活率=[(As-Ab)/(Ac-Ab)]x100%
抑制率=[(Ac-As)/(Ac-Ab)]x100%
As:实验孔吸光度 (含细胞、培养基、CCK-8溶液和药物溶液);
Ac:对照孔吸光度 (含细胞、培养基、CCK-8溶液，不含药物);
Ab:空白孔吸光度 (含培养基、CCK-8溶液，不含细胞、药物)。
注意事项：
1. 未使用完的试剂务必在4℃以下低温保存，拆封后在-20°C避光保存两年，尽量避免反复解冻；
2. CCK-8的培养时间一般为1-4小时，但在培养30分钟左右即可取出肉眼观察显色程度，根据细胞种类而定，需要摸索条件，CCK-8 的最佳反应时间以具体显色的最佳时间为准。建议先做几个孔摸索接种细胞的数量和加入CCK-8试剂后的培养时间 ；
3. 本试剂盒中的WST-8会与还原剂（例如一些抗氧化剂) 反应，从而干扰检测。在细胞增殖-毒性检测前可通过检查背景OD 来确认待检测物质中是否有还原剂。如需去除还原剂影响，可在加CCK-8之前更换新鲜培养基 (除去培养基, 并用培养基洗涤细胞两次，然后加入新的培养基)；
4. 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.
5. 建议采用多通道移液器，以减小平行孔间的差异。加入CCK-8试剂时，建议斜贴着培养板壁加，不要插到培养基液面下加，容易产生气泡，会干扰OD测定；
6. 加入药物中如含有金属，对CCK-8显色有影响。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% 抑制显色反应；
7. 使用96孔板进行检测时，如果细胞培养时间较长，要注意蒸发问题。一方面，由于 96孔板周围一圈最容易蒸发，可以采取弃用周围一圈的办法，改加相同量的PBS、水或培养液;另一方面，可以把96孔板置于靠近培养箱内水源的地方，以缓解蒸发；
8. 当使用标准96孔板时，贴壁细胞的最小接种量至少为1,000个/孔 (100 μl培养基)。检测白细胞时的灵敏度相对较低，因此推荐接种量不低于2,500个/孔 (100 μl培养基)。如果要使用24孔板或6孔板实验，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；
9. 细胞培养时间依细胞的种类和数量（每孔）而不同，通常白细胞显色较弱，需要较长的培养时间（4小时）和大量细胞（~105细胞/孔)；
10. CCK-8试剂对细胞的毒性非常低。它和活细胞内的脱氢酶持续反应使溶液颜色不断加深，OD值不断增加。以下方法可以终止CCK-8反应(96孔板): a) 在显色反应后，将培养板放置4°C冰箱内；b) 每孔加10μL 0.1MHCl溶液; c) 每孔加10 μL 1%(w/v)的SDS (十二烷基硫酸钠) 溶液。反应停止后，应在24小时内测定OD值；
11. 要测定细胞的具体数量，建议同时做标准曲线。
Cell viability and cytotoxicity assays are usually used for drug screening and compound cytotoxicity testing. The CCK-8 kit uses highly water-soluble tetrazolium salt ( called WST-8 ) to produce water-soluble WST-8 for cell proliferation and cytotoxicity assays. Unlike MTT, WST-8 and WST-8 have no cytotoxicity in cell culture medium, so multiple downstream experiments can be performed using the same detection plate. CCK-8 method is a convenient colorimetric method for the determination of cell viability. It does not need the solubilization process and only needs the least steps to provide the results. The CCK-8 method can be used for the determination of 96-well microplates and high-throughput screening of 384-well microplates.
Advantage：
At present, the commercially available liquid CCK-8 kits generally have defects such as harsh storage conditions ( -4C or -20 ), unstable use in different pH ranges, and easy deterioration ( discoloration or precipitation ). The solid instant CCK-8 kit adopts a new formula and Swiss process, which overcomes these shortcomings of the liquid CCK-8 kit. It can be stored at room temperature for a long time ( > 3 years ), ready to use, stable in a wide pH range, and the experimental results are more reliable. Compared with the liquid CCK-8 kit, the solid-soluble CCK-8 kit has higher sensitivity and the biological response time is shortened by half.
Application scope:
It can be used for drug screening, cell proliferation assay, cytotoxicity assay, tumor drug sensitivity test and activity detection of biological factors.
Operating instructions:
This reagent kit can be used for drug screening, cell proliferation assay, cytotoxicity assay, tumor drug sensitivity assay, and activity detection of biological factors.
1. Carefully and slowly tear along the gap in the packaging bag;
2. Pour all the powder in the bag into a clean container containing 10mL of ultrapure water, shake continuously for 1 minute, and use it when the solid is completely dissolved;
3. Unused reagents must be stored at low temperatures below 4 ℃.
Equipment required for testing:
Enzyme reader 96 well plate with 450-490 nm filter;
Carbon dioxide incubator;
96 well plate, sterilized transparent plate for cell detection;
Multi channel pipette (8 or 12 channels: 10-100 µ l);
Blood cell counter or cell counter.
Cell viability testing:
1. Inoculate cell suspension (100 μ l/well) into a 96 well plate and pre culture the plate in a carbon dioxide incubator for 24 hours (37 ℃, 5% CO2);
2. Add 10 μ l of CCK-8 solution to each well (be careful not to generate bubbles in the well as it may affect the reading of OD value);
3. Incubate the culture plate in the incubator for 1-4 hours;
4. Measure the absorbance at 450 nm using an enzyme-linked immunosorbent assay (ELISA) reader;
5. If the OD value is not determined temporarily, 10 μ l of 0.1M HCI solution or 1% w/v SDS solution can be added to each well, and the culture plate can be covered and stored in the dark at room temperature. Within 24 hours of measurement, the absorbance will not change.
Cell proliferation toxicity testing:
1. Inoculate cell suspension (100 μ l/well) into a 96 well plate and pre culture the plate in an incubator for 24 hours (37 ℃, 5% CO2);
2. Add 10ul of different concentrations of the substance to be tested to the culture plate;
3. Incubate the culture plate in the incubator for an appropriate period of time (e.g. 6, 12, 24, or 48 hours);
4. Add 10 μ l of CCK-8 solution to each well (be careful not to generate bubbles in the well as they may affect the reading of the OD value);
5. Incubate the culture plate in the incubator for 1-4 hours;
6. Measure the absorbance at 450nm using an enzyme-linked immunosorbent assay (ELISA) reader;
7. If the OD value is not determined temporarily, 10 μ l of 0.1M HCI solution or 1% w/v SDS solution can be added to each well, and the culture plate can be covered and stored in the dark at room temperature. Within 24 hours of measurement, the absorbance will not change.
Calculation method for cell survival rate/inhibition rate:
Cell survival rate=[As Ab)/(Ac Ab)] x 100%
Inhibition rate=[(Ac As)/(Ac Ab)] x 100%
As: absorbance of experimental wells (including cells, culture medium, CCK-8 solution, and drug solution);
Ac: absorbance of control wells (including cells, culture medium, CCK-8 solution, without drugs);
Ab: Blank well absorbance (including culture medium and CCK-8 solution, excluding cells and drugs).
Points for attention：
1.Unused reagents must be stored at low temperature below 4 °C, and stored in the dark at-20 °C for two years after unpacking, so as to avoid repeated thawing ;
2.The culture time of CCK-8 is generally 1-4 hours, but the naked eye can be taken out to observe the color degree in about 30 minutes. According to the cell type, the conditions need to be explored. The best reaction time of CCK-8 is based on the best time of specific color development.3. It is recommended to do a few holes to explore the number of inoculated cells and the culture time after adding CCK-8 reagent ;
3.The WST-8 in this kit will react with reducing agents ( such as some antioxidants ) to interfere with the detection. Before the cell proliferation-toxicity test, the background OD can be checked to confirm whether there is a reducing agent in the substance to be tested. If the effect of reducing agent needs to be removed, the fresh medium can be replaced before adding CCK-8 ( remove the medium, wash the cells twice with the medium, and then add the new medium ) ;
4.Phenol red in the medium does not affect the experimental results, and the absorbance of phenol red can be eliminated by deducting the absorbance of the background in the blank hole during calculation, so it will not affect the detection.
5.It is recommended to use a multi-channel pipette to reduce the difference between parallel holes. When adding CCK-8 reagent, it is recommended to add it obliquely to the wall of the culture plate, not to insert it under the liquid surface of the medium, which is easy to produce bubbles and interfere with OD determination.
6.If the drug contains metal, it has an effect on the color of CCK-8. The final concentration of 1mM lead chloride, ferric chloride and copper sulfate will inhibit the color reaction of 5 %, 15 % and 90 %, and reduce the sensitivity. If the final concentration is 10mM, the color reaction will be 100 % inhibited ;
7.When using a 96-well plate for detection, if the cell culture time is long, attention should be paid to the evaporation problem. On the one hand, because a circle around the 96-well plate is the easiest to evaporate, the method of discarding the surrounding circle can be adopted, and the same amount of PBS, water or culture medium can be added. On the other hand, the 96-well plate can be placed near the water source in the incubator to alleviate evaporation ; 8.When using standard 96-well plates, the minimum inoculation amount of adherent cells is at least 1,000 cells / well ( 100μl medium ). The sensitivity of detecting white blood cells is relatively low, so it is recommended that the inoculation amount should not be less than 2,500 cells / well ( 100 μl medium ). If you want to use a 24-well plate or a 6-well plate experiment, first calculate the corresponding inoculation amount per well, and add the CCK-8 solution according to 10 % of the total volume of the medium per well ;
9.Cell culture time varies according to the type and number of cells ( per well ), usually the color of white blood cells is weak, requiring a longer culture time ( 4 hours ) and a large number of cells ( ~ 105 cells / well ) ;
10.CCK-8 reagent is very low toxic to cells. The continuous reaction between it and dehydrogenase in living cells makes the color of the solution deepen and the OD value increase. The following methods can terminate the CCK-8 reaction ( 96-well plate ) : a ) After the color reaction, the culture plate was placed in a refrigerator at 4 ° C ; b ) 10μL 0.1MHCL solution was added to each well ; c ) 10 μL 1 % ( w / v ) SDS ( sodium dodecyl sulfate ) solution was added to each well. After the reaction stopped, the OD value should be measured within 24 hours.
11.To determine the specific number of cells, it is recommended to do the standard curve at the same time.
规格
英文名称
Solid instant dissolution Cell Counting Kit-8
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