Cell Counting Kit-8 (CCK-8)

The Selleck Cell Counting Kit-8 (CCK-8) is a ready-to-use one-bottle solution which offers a simple, rapid, reliable and sensitive measurement of cell viability and cytotoxicity of various chemicals. Moreover, Cell Counting Kit-8 (CCK-8) is very safe, fast, nonradioactive and cost-effective compared to other conventional methods such as [3H]-thymidine incorporation assay.

Cell Counting Kit-8 (CCK-8) is a redox indicator that utilizes the highly water-soluble tetrazolium salt WST®-8 [2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt] to produce a water-soluble formazan dye upon reduction by cellular dehydrogenases in the presence of an electron carrier 1-Methoxy PMS in the culture medium (Figure 1). The amount of the formazan dye generated by the activity of dehydrogenases in cells is directly proportional to the number of living cells (Figure 2). The detection sensitivity of Cell Counting Kit-8 (CCK-8) is higher than other tetrazolium salts such as MTT, XTT, MTS or WST-1.

Figure 1: Working mechanisms of Cell Counting Kit-8 (CCK-8).

Figure 2: Cell number vs OD value using Cell Counting Kit-8 (CCK-8). K562 and Hela cells were seeded into 96-well plate at different density in 100 μl culture medium in triplicate respectively. 16 hours later, 10 μl/well Cell Counting Kit-8 (CCK-8) was added, allowing to continuously culture for indicated duration in cell culture incubator. The absorbance at 450 nm were then measured in a microplate reader. Medium only wells were served as blank control. Note the great linear relationship between cell number per well versus OD450 values at all incubation time.

Figure 3: Time dependent color development using Cell Counting Kit-8 (CCK-8). HEK293 cells were seeded into 96-well plate at different concentrations in 100 μl culture medium in triplicate. 16 hours later, 10 μl/well Cell Counting Kit-8 (CCK-8) was added, allowing cells to continuously culture for indicated duration in cell culture incubator. Absorbance at 450 nm was then measured in a microplate reader. Note the highly linear relationship of color development versus incubation time at all cell concentrations.

Required Materials (not included)

• microplate reader (450 nm filter)
• 10 μl, 100-200 μl and multi-channel pipettes.
• 1 % SDS (optional).

Experiment Procedure

1) Inoculate cells in a cell culture flask or dish, and allow cells to adhere or grow for approximately 4–24 hours before proceeding with the assay.
2) Add 1/10 volume of Cell Counting Kit-8 (CCK-8) directly to cells in culture medium. Mix thoroughly to achieve a homogenous solution by lightly tapping the outside of the plate several times while avoiding bubbles. For 96-well plate, add 10 µl Cell Counting Kit-8 (CCK-8) per 100 µl culture medium.
3) Incubate in a cell culture incubator for 0.5 to 4 hours at 37°C until the color turns orange. Over incubation will give false results.
Note: Sensitivity of detection increases with longer incubation times. For samples with fewer cells, use a longer incubation time of up to 24 hours.
4) Record results using microplate reader to measure the absorbance of Cell Counting Kit-8 (CCK-8) at 450 nm. Recommended OD values range between 0.8-1.5, however values between 0.5-2.5 are acceptable.
Note: Slight spontaneous absorbance around 450 nm occurs in culture medium incubated with Cell Counting Kit-8 (CCK-8). This background absorbance depends on the culture medium, pH, incubation time and length of exposure to light. Typical background absorbance after 2 hours incubation is 0.1 - 0.2 absorbance units. To correct for this, prepare one or more control wells without cells, and subtract the average absorbance of the control wells from that of the other wells.
5) Optional: Add 10 μl of 1 % SDS (dissolve 0.1 g SDS with PBS buffer to prepare 10 ml solution) directly to 100 μl of cells to stop the reaction. Signals can be read within 3 days without affecting the absorbance values.
6) Calculation of cell viability:
Cell viability (%) = [A (Drug+) – A (Black)] / [A (Drug-) – A (Black)] x 100%
A (Drug+) : OD value of wells with cells, CCK-8 and drugs;
A (Drug-) : OD value of wells with cells, CCK-8, but without drugs;
A (Black): OD value of wells with culture medium and CCK-8, but without cells.

Brief Summary of the protocol

Applications

1) Cell proliferation determinations—the Selleck Cell Counting Kit-8 (CCK-8) is water soluble, stable in culture, and non-toxic.
2) Cell viability assays—metabolic activity and dye generation changes in proportion to altered viability.
3) Cytokine assays—measure cytokine-induced proliferation. Cells can be recovered and expanded at the end of the study if desired.
4) Cytotoxicity assays—Cells death from cytotoxic chemicals has no effects on color development, only living cells convert the reagent into a colorimetric indicator. The reagent itself has negligible toxicity, and is generally safe for cells.

Note

1. Cell Counting Kit-8 (CCK-8) is ready-to-use solution. Mix the reagent to ensure a homogenous solution before use.
2. If you plan to use longer incubation time (overnight), be sure to maintain sterile conditions during reagent addition and incubation to avoid microbial contaminants. Contaminated cultures will yield erroneous results as microbial contaminants also reduce the Cell Counting Kit-8 (CCK-8).
3. Be sure to include appropriate assay controls. To minimize experimental errors, we recommend making measurements from a minimum of 3–8 replicates of experimental and medium only control.
4. This product is for R&D use only, not for medical, household, or other uses.

• Manual
• SDS