Protocol for Cell Viability Assays: CCK-8 and MTT

Cell viability assays are essential tools in cellular biology for evaluating the effects of drugs, toxins, or other treatments on cell health and proliferation. Two commonly used methods are the CCK-8 (Cell Counting Kit-8) and MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assays. These assays are based on the ability of living cells to reduce tetrazolium salts to formazan, which can be quantified to assess cell viability.

The CCK-8 assay is a colorimetric assay that utilizes a water-soluble tetrazolium salt, WST-8, which is reduced to a colored formazan product by dehydrogenases in viable cells. This method is favored for its simplicity and the ability to measure cell viability in a straightforward, non-toxic manner.

The MTT assay involves the reduction of MTT, a yellow tetrazolium salt, to purple formazan crystals by mitochondrial dehydrogenases in living cells. After solubilization, the intensity of the purple color is proportional to the number of viable cells. Despite its robustness, the MTT assay requires a solubilization step and is less compatible with high-throughput screening compared to CCK-8.

I. Experimental Preparation

1.1 Choosing Appropriate Cell Seeding Density

Adherent Cells: Adjust the cell density based on cell size and experimental requirements.

Suspension Cells: Typically seed 10,000 cells per well in a 96-well plate. Use a cell counter for accurate seeding.

Note: Use healthy cells for seeding. Avoid using cells that have undergone too many passages. A seeding density that is too low may result in less noticeable drug effects, while too high a density may cause cell death. Recommended seeding density is 10,000 cells per well, 100 µL per well.

1.2 Setting Drug Concentration and Treatment Duration

Determine drug concentration based on the sensitivity of the cell type. Review the literature to establish an initial range, calculate the IC50 value, and adjust the drug concentration accordingly.

1.3 Culturing Time

If the culture period exceeds 48 hours, change the culture medium to maintain cell health.

1.4 Aseptic Technique

Ensure aseptic conditions during MTT or CCK-8 assays to prevent contamination, which can affect absorbance accuracy. Bacterial contamination can increase optical density and interfere with results.

1.5 Setting Controls

Blank Control: Add medium, MTT or CCK-8, and DMSO.

Control Wells: Add cells, medium, and MTT or CCK-8. Include DMSO for drug solubilization.

Treatment Wells: Add cells, medium, MTT or CCK-8, and drugs at various concentrations.

1.6 Setting Replicates

Prepare at least 3 replicates per treatment group, preferably 3-5, to ensure reliability.

II. Experimental Procedure

2.1 Cell Seeding

Collect cells at the logarithmic growth phase and adjust the cell suspension to 10,000 to 20,000 cells/mL. Add 100 µL of this suspension to each well of a 96-well plate. Fill edge wells with sterile PBS or an appropriate medium to prevent evaporation. Prepare 3 to 5 replicates per well.

2.2 Drug Treatment

After seeding, incubate the plate at 37°C with 5% CO2 for 1 hour. Add drugs prepared fresh using a dilution method in the culture medium. Use guidelines for drug solubilization as specified (e.g., use DMSO for long-term storage solutions).

2.3 Incubation

Incubate the treated plate at 37°C with 5% CO2 until the end of the drug treatment period.

2.4 Adding MTT or CCK-8

MTT: Add 10 µL of MTT solution (5 mg/mL, 0.5% MTT) to each well and incubate for 4 hours.

CCK-8: Add 10 µL of CCK-8 reagent to each well and incubate for 1-4 hours.

Note: Prepare the MTT solution fresh and filter it. Store at 4°C in the dark. Maintain sterility as MTT is sensitive to bacterial contamination. Use PBS for dissolution and assist with a 60°C water bath and ultrasonic treatment for complete dissolution.

2.5 Measuring Absorbance

MTT: After incubation, add 100 µL of MTT solubilization solution to each well. Allow sufficient time for crystals to dissolve. Measure the absorbance at 570 nm (with 630 nm reference) using a microplate reader.

CCK-8: Measure the absorbance at 450 nm with a reference wavelength set above 600 nm.

III. Other Notes

3.1 Cell Density Control: For suspension cells, aim for approximately 10,000 cells per well. Avoid excessive density, especially with tumor cells, to ensure accurate and reliable results.

3.2 Experimental Variability: MTT and CCK-8 assays are relatively coarse methods, so minor fluctuations or errors in results are normal.

3.3 Mixing Cell Suspension: Ensure thorough mixing of cell suspension. If left standing for a long time, gently pipette to mix. Uneven cell distribution can lead to inconsistent results. Minimize blowing and pipetting errors to preserve cell viability.

3.4 Suspension Volume Control: Keep the volume of cell suspension around 6-7 mL to avoid contamination and bubble formation.

3.5 Pipetting Technique: Place the pipette tip at the bottom of the suspension for aspiration and near the liquid surface for dispensing to avoid bubbles.

3.6 Plate Handling: After adding to the plate, gently shake to ensure even cell distribution. After seeding, let the plate sit for 2 minutes and check for uniform cell distribution.

3.7 Edge Effect: Use the edge wells of a 96-well plate only for blanks to avoid cell culture. Fill these wells with water, PBS, or medium to prevent evaporation.

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