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Detailed Protocol for CCK-8 Assay
01 Basic Experiment: CCK8 Assay

Cell Counting Kit-8 (CCK-8) is a colorimetric assay kit widely used for the rapid, highly sensitive, and non-radioactive detection of cell proliferation and cytotoxicity, based on WST-8. The CCK-8 solution can be directly added to cell samples without the need for pre-preparation of various components. In the presence of electron coupling reagents, WST-8 can be reduced by certain dehydrogenases in the mitochondria to generate an orange-yellow formazan (Figure 1). The more and faster the cells proliferate, the darker the color; the greater the cytotoxicity, the lighter the color. For the same type of cells, there is a linear relationship between the color intensity (amount of formazan produced) and the cell number (Figure 2).

Figure 1. Structural formula of WST-8 and WST-8 formazan.
Figure 2. Schematic diagram of CCK-8.

WST-8 is an upgraded alternative to MTT and has significant advantages over MTT and other similar products such as XTT and MTS:

1.The formazan generated by MTT, reduced by mitochondrial dehydrogenases, is not water-soluble and requires specific solvents for dissolution; in contrast, the formazan produced by WST-8 and by XTT and MTS is water-soluble, eliminating the need for subsequent dissolution steps.

2.The formazan produced by WST-8 is more easily soluble than that produced by XTT and MTS.

3.WST-8 is more stable than XTT and MTS, making experimental results more reliable.

4.WST-8 has a wider linear range compared to MTT, XTT, and MTS, higher sensitivity, and greater stability.

WST-8 exhibits no significant toxicity to cells. After adding the CCK-8 solutions, the color can be read repeatedly at different times using a microplate reader, allowing for greater flexibility in determining the optimal measurement time.

02 Detailed Protocols

The MCE CCK8 Assay Kit (HY-K0301) can be used to detect cell proliferation induced by cytokines and to assess cytotoxicity induced by anticancer drugs or other toxic agents, as well as to evaluate drug-induced growth inhibition. Let's explore the specific procedures of the CCK8 assay using HY-K0301 as an example!

Figure 3. Flowchart of CCK8 assay.

1. Creating a Standard Curve

(1)Cell Counting: First, use a cell counting chamber to count the number of cells in the prepared cell suspension, then seed the cells.

(2)Dilution: Dilute the cells in culture medium to create a cell concentration gradient, typically making 5-7 different concentrations, with 4-6 replicates for each concentration.

(3)Incubation: After seeding, incubate for 2-4 hours to allow the cells to adhere. Then, add 10 μL of the CCK-8 reagent to each well containing 100 μL of culture medium and incubate for a specific time before measuring the optical density (OD). This will create a standard curve with cell number on the x-axis and OD value on the y-axis. The cell number of unknown samples can be determined using this standard curve.

Note: The use of this standard curve requires that experimental conditions be identical.

2. Cell Viability Detection

(1)Cell Seeding: Seed the cell suspension (100 μL/well) in a 96-well plate and incubate the plate in an incubator for 24 hours.

(2)Adding CCK-8 Solution: Add 10 μL of CCK-8 solution to each well (taking care to avoid bubbles).

(3)Incubation: Incubate the plate in the incubator for 1-4 hours.

(4)OD Measurement: Measure the absorbance at 450 nm using a microplate reader.

3. Cell Proliferation and Toxicity Detection

(1)Cell Seeding: Seed the cell suspension (100 μL/well) in a 96-well plate and incubate the plate in an incubator for 24 hours.

(2)Drug Addition: Add different concentrations of the test drug to the wells.

(3)Incubation: Incubate the plate in the incubator for an appropriate period.

(4)Adding CCK-8 Solution: Add 10 μL of CCK-8 solution to each well (taking care to avoid bubbles).

(5)Incubation: Incubate the plate in the incubator for 1-4 hours.

(6)OD Measurement: Measure the absorbance at 450 nm using a microplate reader.

4. Calculation Formulas

(1)Cell Viability Rate = [(As-Ab) / (Ac-Ab)] x 100%

(2)Inhibition Rate = [(Ac-As) / (Ac-Ab)] x 100%

As: Absorbance of the experimental wells (containing cells, culture medium, CCK-8 solution, and drug solution).

Ac: Absorbance of the control wells (containing cells, culture medium, CCK-8 solution, without drug).

Ab: Absorbance of the blank wells (containing culture medium and CCK-8 solution, without cells or drug).

5. MCE Verification

Figure 4. CCK-8 kit to detect cell activity. Cell line: HEK293; culture medium: DMEM, 10% FBS; culture conditions: 37°C, 5% CO2, 2 h.
Figure 5. CCK-8 kit to detect DDP cytotoxicity. Cell lines: U87, SK-HEP1; culture medium: DMEM, 10% FBS; chemical drugs: 200 μM Cisplatin (DDP); culture conditions: 37°C, 5% CO2, 2 h.

Note: If the test drug has oxidative or reductive properties, it is recommended to change to fresh culture medium before adding CCK-8 to eliminate the drug's influence. If the impact of the test drug is minimal, you can skip the medium replacement and directly subtract the blank absorbance after adding the test drug to the culture medium.

03 Practical cases

This time, we will look at the basic application of the CCK-8 assay based on the MCE customer's published literature: "RNA interference-mediated depletion of TRPM8 enhances the efficacy of epirubicin chemotherapy in prostate cancer LNCaP and PC3 cells[1].

 

In this study, cell growth and viability were measured using the cell proliferation and cytotoxicity reagent CCK-8 (HY-K0301). The cell viability index in this study was calculated based on optical density (OD) with the formula: experimental OD value / control OD value × 100%. The experiments were repeated three times.

Experimental Groups

siTRPM8: Cells transfected with siTRPM8

siCON: Cells transfected with siCON (control)

Parental: Untransfected cells, referred to as parental

SB: p38 inhibitor, SB203580

SP: JNK inhibitor, SP600125

Experimental Cells: Prostate cancer cell lines LNCaP or PC3 cells

Case 1

To assess the impact of TRPM8 knockdown (siRNA) on the proliferation of LNCaP and PC3 cells using the CCK-8 assay.

Protocol

Seed both transfected and untransfected LNCaP or PC3 cells (5x10³ cells per well) in a 96-well plate, with ten wells for each group. Add fresh culture medium and incubate for an appropriate period. Then, add 10 µL of CCK-8 working solution, and incubate the mixture at 37°C for 4 hours. Measure the absorbance using a microplate reader.

Conclusion

TRPM8 knockdown inhibited the growth of LNCaP and PC3 cells. Compared to parental and siCON cells (negative control), the growth of siTRPM8 cells was significantly suppressed on day 2 (Figure 6).

Figure 6. The effect of TRPM8 knockdown on the proliferation of (A) LNCaP and (B) PC3 cells was measured by CCK-8 assay[1].

Case 2

To evaluate the effect of siTRPM8 on the sensitivity to EPI (Epirubicin) using the CCK-8 drug sensitivity assay.

Protocol

To assess chemoresistance, cells were first incubated for 12 hours to allow attachment. Then, the cells were incubated with the vector (0.01% DMSO) or different concentrations (0, 200, 400, 600, 800, 1,000 ng/mL) of EPI for 48 hours, followed by CCK-8 measurement. Results were expressed as a percentage relative to the control group, which was set at 100%, with the control treated with the vector (0.01% DMSO). Optical density was read using a microplate reader.

Conclusion

TRPM8 knockdown enhanced EPI-induced growth inhibition. Compared to parental and siCON cells, siTRPM8 cells exhibited significantly decreased viability after 48 hours of incubation with the indicated concentrations of EPI, in a dose-dependent manner (Figure 7). When treated with 600 µM EPI, the viability of siTRPM8 cells was markedly lower than that of parental and siCON cells.

Figure 7. TRPM8 knockdown significantly enhanced EPI-induced (A) LNCaP and (B) PC3 cell viability inhibition[1].

Case 3

To analyze the effects of the p38 inhibitor (SB203580, 20 µM) and JNK inhibitor (SP600125, 10 µM) on EPI-mediated proliferation inhibition in siTRPM8 cells using the CCK-8 assay.

Protocol

To verify the role of the MAPK signaling pathway, add the p38 inhibitor SB203580 (20 µM) and SP600125 (10 µM) to the culture medium 2 hours prior to the addition of EPI. Cell viability was assessed using the Cell Counting Kit-8.

Conclusion

Specific inhibitors of p38 and JNK diminished the enhancement of EPI sensitivity induced by siTRPM8 (Figure 8).

Figure 8. Specific inhibitors of p38 and JNK reduced the enhancement of EPI sensitivity induced by siTRPM8[1].

Product Recommendation

Cell Counting Kit-8

Also known as the CCK-8 kit, this is a rapid and highly sensitive kit for detecting cell viability and cytotoxicity, based on WST-8.

Epirubicin

Epirubicin (4'-Epidoxorubicin) is a semi-synthetic derivative of doxorubicin, which inhibits topoisomerase and exhibits antitumor activity. It interferes with DNA and RNA synthesis and acts as an inhibitor of Forkhead box protein p3 (Foxp3), suppressing regulatory T cell activity.

Adezmapimod

Adezmapimod (SB 203580) is a selective, ATP-competitive p38 MAPK inhibitor, with IC50 values of 50 nM for SAPK2a/p38 and 500 nM for SAPK2b/p38β2.

SP600125

SP600125 is an orally bioavailable, reversible, ATP-competitive JNK inhibitor, with IC50 values of 40 nM for JNK1 and JNK2, and 90 nM for JNK3.