Ultra-Sensitive Cell Proliferation and Cytotoxicity Assay Kit (CCK-8)
Results in 30 minutes; even small cell samples can be tested.
One-click copy of information
Instruction manualPrice:
¥ 300
Item Number:
100-120
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Specification
- 500T
- 1000T
- 10000T
隐藏域元素占位
- Product Description
- Composition
- Precautions
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Product Storage:
Store at 4°C away from light; shelf life: one year. Store at −20°C; shelf life: two years. Avoid repeated freeze–thaw cycles. Transport with ice packs.
Product Overview:
Cell Counting Kit‑8 (CCK‑8) is a reagent kit based on WST‑8, widely used for rapid, highly sensitive assays of cell proliferation and cytotoxicity. The CCK‑8 reagent contains WST‑8 (chemical name: 2-(2‑methoxy‑4‑nitrophenyl)-3-(4‑nitrophenyl)-5-(2,4‑disulfophenyl)-2H‑tetrazolium monosodium salt), a compound similar to MTT. In the presence of the electron‑coupling agent 1‑methoxy‑5‑methylphenazinium methyl sulfate (1‑Methoxy PMS), WST‑8 can be reduced by mitochondrial dehydrogenases to formazan, a highly water‑soluble orange‑yellow product. The amount of formazan produced is directly proportional to the number of viable cells; thus, this property enables direct quantification of cell proliferation and cytotoxicity: the greater and faster the cell proliferation, the deeper the color, while increased cytotoxicity results in a lighter color.
This kit is an ultra-sensitive CCK‑8 assay that has been optimized to significantly shorten the incubation time; typically, a 0.5–1 hour incubation is sufficient to obtain optimal signal intensity. However, depending on experimental conditions such as cell type and cell density, the required incubation time may vary.
Instructions for Use
Prepare a standard curve (when determining the exact cell number)
1. Prepare a cell suspension: perform cell counting.
2. Seed into a 96-well plate: Serially dilute the cell suspension with culture medium at equal ratios (e.g., 1:2) to generate a series of cell concentration gradients. Typically, prepare 3–5 concentration gradients, with 3–6 replicates per group. Add approximately 100 μL of cell suspension to each well.
3. Incubation in a 37°C incubator: After seeding, cells typically require 2–4 hours to adhere to the culture surface; if adhesion is not required, this step may be omitted.
4. Add 10 μL of the ultra-sensitive CCK‑8 solution to each well: Since the volume of CCK‑8 added to each well is relatively small, residual reagent adhering to the well walls may introduce variability; therefore, we recommend gently tapping the microplate after adding the reagent to facilitate thorough mixing. Alternatively, you may prepare culture medium containing 10% CCK‑8 directly.
Add by replacing the liquid. Be careful to avoid bubble formation in the wells, as this may interfere with absorbance measurements.
5. After incubating in the incubator for 0.5–3 hours, measure the absorbance at 450 nm and construct a standard curve with cell number on the x-axis and absorbance on the y-axis. This standard curve can then be used to determine the cell number in unknown samples. (Note that this standard curve is valid only if experimental conditions remain consistent, allowing for accurate determination of the initial cell seeding density and the appropriate incubation time following addition of CCK‑8.)
Cell viability assay
1. Prepare a cell suspension: perform cell counting.
2. Seed an appropriate number of cells into a 96-well plate, adding approximately 100 μL of cell suspension per well; three replicates may be included.
3. Incubation in a 37°C incubator: After seeding, cells typically require 2–4 hours to adhere to the culture surface; if adhesion is not required, this step may be omitted.
4. Add 10 μL of the ultra-sensitive CCK‑8 solution to each well: Since the volume of CCK‑8 added to each well is relatively small, residual reagent adhering to the well walls may introduce variability; therefore, we recommend gently tapping the microplate after adding the reagent to facilitate thorough mixing. Alternatively, you may prepare culture medium containing 10% CCK‑8 directly.
Add by replacing the liquid. Be careful to avoid bubble formation in the wells, as this may interfere with absorbance measurements.
5. Incubate in the incubator for 0.5–3 hours: The amount of formazan produced varies depending on the cell type; in most cases, a 1-hour incubation is sufficient. If the color development is insufficient, you may extend the incubation to determine the optimal conditions, particularly for cells derived from blood.
Formazan is produced in very small amounts and requires a relatively long color-development time.
6. Measure the absorbance at 450 nm: If you do not measure the absorbance immediately, add 10 μL of a self-prepared 0.1 M HCl solution or a 1% w/v SDS solution to each well, then cover the microplate and store it protected from light at room temperature; the absorbance will remain stable for up to 24 hours.
Cell Proliferation–Cytotoxicity Assay
1. Prepare a cell suspension: perform cell counting.
2. Seed an appropriate number of cells into a 96-well plate, adding approximately 100 μL of cell suspension per well; three replicates are recommended.
3. Incubation in a 37°C incubator: After seeding, cells typically require 2–4 hours to adhere to the culture surface; if adhesion is not required, this step may be omitted. Alternatively, the incubation time can be adjusted according to experimental needs.
4. Add 0–10 μL of the test compound at different concentrations to each well.
5. Incubation in a 37°C incubator: The duration of drug exposure should be determined based on the properties of the compound and the cells’ sensitivity, typically aligned with the cell cycle; in general, it should span at least one full cell cycle.
6. Add 10 μL of the ultra-sensitive CCK‑8 solution to each well: Since the volume of CCK‑8 added to each well is relatively small, residual reagent adhering to the well walls may introduce variability. It is recommended to gently tap the microplate after adding the reagent to facilitate thorough mixing. Alternatively, you can directly prepare culture medium containing 10% CCK‑8.
Add the reagent by replacing the culture medium. Be careful to avoid bubble formation in the wells, as this may interfere with absorbance measurements. (Note: If the analyte is oxidizing or reducing, remove the original culture medium before adding CCK‑8, wash the cells twice with fresh medium, and then add fresh medium to eliminate any drug‑related interference. If the drug effect is minimal, you may skip medium replacement and simply subtract the blank absorbance measured in the medium containing the drug.)
7. Incubate in the incubator for 0.5–3 hours: The amount of formazan produced varies depending on the cell type; in most cases, a 1-hour incubation is sufficient. If the color development is insufficient, you may extend the incubation to determine the optimal conditions, particularly for cells derived from blood.
Formazan is produced in very small amounts and requires a relatively long color-development time.
8. Measure the absorbance at 450 nm: It is recommended to perform the measurement using a dual-wavelength approach, with a detection wavelength of 450–490 nm and a reference wavelength of 600–650 nm. If the absorbance will not be measured immediately, add 10 μL of a self-prepared 0.1 M HCl solution or a 1% w/v SDS solution to each well.
Cover the microtiter plate and store it protected from light at room temperature; the absorbance will remain stable for up to 24 hours.
Calculation formula
Cell viability = [(As - Ab) / (Ac - Ab)] × 100% Inhibition rate = [(Ac - As) / (Ac - Ab)] × 100%
As: The absorbance of the experimental well (containing cell culture medium, CCK‑8, and the test drug)
Ac: Absorbance of the control well (culture medium containing cells, CCK‑8, and no test compound)
Ab: Absorbance of the blank well (culture medium containing no cells or test compound, plus CCK-8)
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Product Composition
Product item number Product Composition Specifications 100-120-500 CCK-8 Ultra-Sensitive Solution 5mL 100-120-1000 CCK-8 Ultra-Sensitive Solution 5 mL × 2 100-120-10000 CCK-8 Ultra-Sensitive Solution 5 mL × 20 -
Precautions
1. It is recommended to conduct preliminary experiments to optimize the number of cells seeded and the incubation time after adding the CCK‑8 reagent.
2. Determination of the CCK‑8 reaction time: In general, leukocytes are relatively difficult to develop color, so it is necessary to increase the cell number and extend the CCK‑8 reaction time. Compared with adherent cells, suspension cells are more challenging to stain; therefore, after adding CCK‑8, suspension cells should be incubated for 0.5–3 hours,
The plate can first be removed from the incubator, and the degree of color development can be assessed visually or by spectrophotometry. If color development is slow, the cells may be further incubated for several additional hours before evaluation. For adherent cells, the typical CCK‑8 incubation time is 0.5–3 hours; after approximately 20 minutes of incubation, the plate can be removed for a visual assessment of color intensity. Cell seeding density per well: When using a standard 96‑well plate, the minimum seeding density for adherent cells should be at least 1,000 cells per well.
(100 μl of culture medium). The sensitivity for detecting white blood cells is relatively low; therefore, a seeding density of at least 2,500 cells per well (in 100 μl of culture medium) is recommended. If you plan to use a 24-well or 6-well plate, first calculate the appropriate inoculum volume for each well, and add CCK‑8 solution to each well at 10% of the total culture medium volume.
3. Set up a blank control: Add CCK‑8 to cell‑free culture medium, incubate for a specified period, and measure the absorbance at 450 nm; this serves as the blank control. When performing drug‑treatment experiments (cytotoxicity assays), also account for drug absorption by adding CCK‑8 to the drug‑containing culture medium, incubating for a set time, and measuring the absorbance at 450 nm as the blank control.
4. Substances that may interfere with CCK‑8 assays: Since the CCK‑8 assay measures cell viability by detecting dehydrogenase‑catalyzed reactions in living cells, redox‑active compounds present in the sample can potentially skew the results—reducing agents increase absorbance, while oxidizing agents decrease it. Therefore, steps should be taken to eliminate the influence of such substances. Phenol red and serum do not significantly affect the assay; the absorbance attributable to phenol red in the culture medium can be corrected by subtracting the background absorbance measured in wells containing only the culture medium, ensuring that these components do not compromise the assay outcomes.
5. Measurement wavelength: If the sample is a highly turbid cell suspension, it is recommended to perform measurements using a dual-wavelength approach, with a detection wavelength of 450 nm and a reference wavelength of 600–650 nm. If a 450‑nm filter is unavailable, a filter with transmittance in the 430–490 nm range may be used; however, the 450‑nm wavelength provides the highest detection sensitivity.
6. When incubated in a cell culture incubator, the wells in the outermost row of the microplate are most prone to drying and evaporation, which can introduce errors due to volume inaccuracies. In such cases, the wells in the outermost row should be filled only with culture medium and not used for assay measurements.
7. If the cells have been cultured for an extended period and the culture medium has changed color, wash the cells, replace the medium, and then add CCK‑8 for assay.
8. Please wear a lab coat and disposable gloves when performing the procedure.
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