Cell Counting Kit - 8

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This process is incredibly easy to use, with rapid and real-time detection that eliminates the need for medium removal. The low levels of endotoxin make it a more environmentally friendly approach. Additionally, there is no need to incorporate organic solvents such as DMSO, and consumables are minimized.

The broad applications of CCK-8 encompass drug screening, cytotoxicity assay, cell proliferation assay, as well as tumor drug sensitivity tests and detection of biological factor activity. This versatility has made CCK-8 a valuable tool in various fields of research and medication development.

Cell Counting Kit - 8:The Complete Buying Guide

What is CCK-8?

CCK-8, an upgraded version of MTT, offers a convenient solution for cell sample analysis. Unlike MTT, CCK-8 solution can be directly added to the samples without the need for premixing with various components. This feature makes it fast and efficient. The basis of CCK-8 lies in its water-soluble four azole salt WST-8. Within the presence of an electron coupling reagent, the WST-8 can be reduced by the dehydrogenase in the mitochondria, resulting in the production of an orange-yellow dyestuff. This dyestuff can be dissolved in the tissue culture medium and is directly proportional to the number of viable cells. By utilizing colorimetry techniques, the CCK-8 assay allows for the dynamic measurement of viable cells, making it a valuable tool for assessing cell proliferation and drug toxicity. It is commonly employed for examining cell cycle progression and apoptosis in both adherent and suspended cells. However, if used for tissue analysis, the tissue must first be digested into a single cell state before measurement can be obtained.

How many types of CCK-8?

CCK-8 kits only one type, but there are kits that function similarly to CCK-8 like MTT, XTT, MTS, etc. CCK-8 is more sensitive and stable compared to others.

What are the components of CCK-8?

CCK-8 solution, i.e. cell counting kit-8 cell counting reagent, contains WST-8: Chemical Name: 2 - (2-methoxy-4-nitrophenyl) - 3 - (4-nitrophenyl) - 5 - (2,4-benzene disulfonate) - 2H tetrazole monosodium salt, which is reduced to a highly water-soluble yellow methazine product (formazan) by dehydrogenase in cell mitochondria under the action of electron carrier 1-methoxy-5-methylphenazine dimethyl sulfate (1-methoxy PMS), The amount of methyl alcohol produced is proportional to the number of living cells. The absorption value of 450nm can be measured indirectly by enzyme-linked immunosorbent assay (ELISA), which can indirectly reflect the number of living cells. This method has been widely used in the detection of bioactive factors, large-scale screening of anti-tumor drugs, cell proliferation test, cytotoxicity test and drug sensitivity test.

What is the detection range of CCK-8?

The chromogenic darkness of CCK-8 is closely related to the number of viable cells, so its detection range also depends on the number of viable cells. Generally the range of cell number in 100uL cell suspension per well should be no less than 1000 and the highest should be no higher than 107 per well, so its detection range should fall between 103~107.

What are the advantages of CCK-8?

The operation is simple, the detection is fast and real-time, and the operation of removing the medium is eliminated. Endotoxin is low, which is more advantageous to environmental protection. It does not use organic solvents DMSO, and requires less consumables.

What specifications are for CCK-8?

We Absin provide 5ml (500T) and 50ml (5000T) of CCK-8(cat. abs50003).

Why is CCK-8 needed?

Both CCK-8 and MTT methods are suitable for large-scale and rapid evaluation of drugs. However, because of the different ways of cell death caused by drug action, such as cell senescence and autophagy, the sensitivity decreases. The main reason is that these two methods are based on indirect detection of mitochondrial activity in living cells. Aging cells are living cells. Autophagy is also a living cell. Mitochondria remain intact and MTT staining is positive. Therefore, CCK-8 and MTT are interfered by these cells. By prolonging the incubation time to 96h, we can minimize the effect of cell proliferation but not survive. The results of CCK-8 assay show a similar sensitivity to colony formation rate.

How does CCK-8 work?

Cell Counting Kit-8, referred to as CCK-8, is based on WST-8 (chemical name: 2 - (2-methoxy-4-nitrophenyl) - 3 - (4-nitrophenyl) - 5 - (2,4-disulfobenzene) - 2H - tetrazolium monosodium salt). CCK-8 is intended for rapid and highly sensitive detection of cell proliferation and cytotoxicity.

What is the difference between CCK-8 and MTT?

Compared with MTT, CCK-8 has obvious advantages. Within a certain cell number, the linear relationship between CCK-8 and the number of living cells was obviously higher than that of MTT. Moreover, MTT method produces blue purple crystals insoluble in water, which need to be dissolved by organic solvent DMSO. Incomplete dissolution or cell loss often occur during operation, which affects the accuracy of the results. The CCK-8 method is only one step operation of adding dye. The operation is simple and the detection can be real-time, eliminating the operation of removing the culture medium. Environmental protection is more advantageous, and no organic solvent DMSO is needed.

The advantages of each two assay kits are compared as follows:

Does CCK-8 assay need to be protected from light?

There is no need to avoid light operation during normal experiment. If the OD value is not determined temporarily, it is possible to add 10 to each hole. μ L of 0.1 M HCl solution or 1% w / V SDS solution, cover the culture plate and keep it away from light at room temperature. The absorbance will not change within 24 hours.

How to use CCK-8 and what is the protocol ?

CCK-8 were used for drug screening, cell proliferation assays, cytotoxicity assays, and tumor drug susceptibility testing. So what are the operational steps used for cytotoxicity assays?

Method / Step 1: Make up 100ul of the cell suspension in a 96 well plate. Allow the plates to pre incubate for 24 hours in the incubator (at 37℃，5% CO2).

Method / Step 2: Add 10ul of different concentrations of the substance to be tested to the plate.

Method / Step 3: Incubate the plate in the incubator for an appropriate period of time (e.g. 6, 12, 24 or 48 hours).

Method / Step 4: Add 10 ul of CCK8 solution (Engreen) to each well (be careful not to generate air bubbles in the wells so that they will affect the reading of OD values).

Method / Step 5: Incubate the plate within the incubator for 1-4 hours.

Method / Step 6: Determine the absorbance at 450 nm with a microplate reader.

Method / Step 7: If the OD value is not determined for the time being and is intended for later testing, 10ul of 0.1 M HCl solution or 1% w/v SDS solution can be added to each well, and cover the culture plate protected from light then store at room temperature. No change in absorbance occurs over a period of 24 hours.

Are there other kits that can replace CCK-8?

No, CCK-8 kit detection method has its uniqueness and cannot be replaced. After determining the appropriate experimental conditions, CCK-8 method can obviously reduce the interference of aging cells. It is a simple, repeatable and sensitive detection method, which is worthy of further promotion.

Is CCK-8 accurate?

The accuracy of CCK-8 kit is relatively high. Compared with MTT method, the formazan produced by MTT experiment is not water-soluble and needs to be dissolved with DMSO and other organic solvents. However, the formazan produced by CCK-8 method is water-soluble, eliminating the dissolution step, so the error caused by this operation step is reduced; In addition, the CCK-8 method has little cytotoxicity, and can be selected several times to select the best determination time. Compared with the MTT method, the linear range is wider and the sensitivity is higher.

Which supplier’s cck-8 kit can we choose ?

The quality of selecting CCK-8 kit is the primary factor, which can be seen from the citation rate. The higher the citation rate is, the higher the quality of the article is, and the quality of proof can withstand many tests. The second is price, quality and cost reduction. Of course, no matter which supplier to choose, it is best to apply the contrast first and make an objective evaluation.

What’s application of CCK-8 Kit ?

CCK-8 is widely used, such as drug screening, cell proliferation assay, cytotoxicity assay, tumor drug sensitivity test and biological factor activity detection.

CCK-8 assay principle:

WST-8 is an upgraded product of MTT. It can be reduced by the dehydrogenase in the mitochondria to produce highly water-soluble orange yellow product (formazan) when the electron coupling reagent exists. The depth of color is directly proportional to cell proliferation, which is inversely proportional to cytotoxicity. The OD value was measured at 450mM wavelength by enzyme labelling instrument, which indirectly reflected the number of living cells.

Notes for CCK-8 assay

1. It is recommended to do several wells first to check the number of seeded cells and the incubation time after adding CCK-8 reagent.

2. A multichannel pipette is recommended for conditional cases, which can reduce parallel well to well variability. When CCK-8 reagent is added, it is recommended to adhere obliquely to the culture plate wall plus. Do not plug below the liquid surface of the medium for addition, which is easily to generate air bubbles that will interfere with the OD value reading.

3. The white blood cells may need to be cultured for longer periods of time.

4. When using standard 96 well plates, the minimum seeding of adherent cells was at least 1000 cells / well (100μL culture medium). The sensitivity when detecting white blood cells is relatively low, so an inoculum size of no less than 2500 cells / well is recommended (100μL culture medium). If 24-well or 6-well plate experiments are to be used, first calculate the corresponding seeding volume per well and add CCK-8 solution at 10% of the total volume of medium per well.

5. If a 450 nm filter is not available, a filter with absorbance between 430-490 nm can be used. But the 450 nm filter has the highest detection sensitivity.

6. In calculating the absorbance of phenol red in the medium, it can be eliminated by subtracting the absorbance of the background in the blank well, so it will not cause an effect on the detection.

7. For your safety and health, wear lab coat and handle with disposable gloves.

What is the MOQ of Absin CCK-8?

The minimum order is one kit，abs50003-5ml (500T)

Can Absin support CCK-8 samples?

 If you want sample kit, please cotact us info@absin.cn.

How long can Absin CCK-8 be delivered?

Absin CCK-8 has always been in stock. Generally, it can be delivered in 3-5 days in China. The specific delivery time is different from the address of the ordering country.

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