A ready-to-use, one-bottle WST-8 reagent for rapid colorimetric measurement of viable cell number, cell proliferation and compound-induced cytotoxicity. Viable-cell dehydrogenase activity converts WST-8 into a water-soluble orange formazan product that is quantified at 450 nm.
Product overview
The AffiASSAY® WST-8 Cell Viability, Proliferation & Cytotoxicity Assay Kit is designed for convenient, nonradioactive assessment of metabolically active cells in in vitro models. The highly water-soluble tetrazolium salt WST-8 is reduced in viable cells to generate an orange, water-soluble formazan dye. The amount of formazan produced is proportional to the number of living cells within the assay's validated linear range.
Because the reaction product remains soluble in culture medium, no solubilization step is normally required. The one-bottle format supports straightforward addition to multiwell plates and is suitable for routine viability measurements, proliferation experiments and evaluation of treatment-related cytotoxicity.
Key benefits
Ready to use
One-bottle liquid reagent minimizes preparation steps and reduces pipetting variability.
Water-soluble signal
WST-8 generates a soluble orange formazan product, avoiding the crystal-solubilization step required by some tetrazolium assays.
Sensitive colorimetric readout
Quantification at 450 nm provides a practical readout using a standard absorbance microplate reader.
Flexible applications
Supports viable cell counting, proliferation studies, cytotoxicity testing and concentration-response experiments.
Nonradioactive workflow
Measures cellular metabolic activity without radioactive labeling or specialized detection equipment.
Plate-based convenience
Optimized for simple addition to 96-well culture plates with a typical 1-4 hour signal-development period.
How the WST-8 assay works
Viable cells generate reducing equivalents
Metabolically active cells maintain dehydrogenase activity and intracellular NADH-dependent reduction pathways.
WST-8 is reduced
In the presence of an electron mediator, cellular dehydrogenase activity reduces WST-8 to its orange formazan form.
Absorbance reflects viable cell number
The soluble formazan signal is read at 450 nm and increases with the number of viable cells within the assay's working range.
Recommended assay parameters
| Parameter | Recommended starting condition | Practical guidance |
|---|---|---|
| Plate format | 96-well plate | Use consistent well volumes and include appropriate blank and untreated-control wells. |
| Culture volume | 100 µL per well | Maintain the same final volume across samples and controls. |
| WST-8 reagent | 10 µL per well | Add carefully and avoid introducing bubbles, which can interfere with absorbance readings. |
| Color development | 1-4 hours at the cell-culture temperature | Optimize incubation time for each cell type, density and treatment condition. |
| Primary readout | 450 nm | Read using an absorbance microplate reader after gently mixing the plate. |
| Reference readout | 600 nm or higher, when needed | For turbid samples, subtract a reference wavelength reading from the 450 nm measurement. |
| Typical starting density for treatment assays | 104-105 cells/well | Perform a cell-number titration because the optimal density varies considerably by cell line. |
General protocols
A. Viable cell-number determination
- Dispense 100 µL of cell suspension into each well of a 96-well plate.
- Pre-incubate the plate under the appropriate culture conditions, such as 37°C and 5% CO2 for mammalian cells.
- Add 10 µL of WST-8 reagent to each well without creating bubbles.
- Incubate for 1-4 hours under standard culture conditions.
- Gently mix and measure absorbance at 450 nm using a microplate reader.
- Subtract the blank value and use a cell-number standard curve when quantitative cell-number estimation is required.
B. Cell proliferation or cytotoxicity assay
- Seed cells in 100 µL of culture medium per well. A starting range of 104-105 cells/well may be evaluated during optimization.
- Allow cells to recover or attach as required by the experimental model.
- Add test compounds, vehicle controls and untreated controls at the desired concentrations.
- Incubate for the selected treatment period, for example 6, 12, 24 or 48 hours.
- Add 10 µL of WST-8 reagent to each well.
- Incubate for 1-4 hours, gently mix the plate and read absorbance at 450 nm.
Data calculation and interpretation
Blank correction
Subtract the absorbance of medium-plus-reagent blank wells from every experimental well.
Relative cell viability
Relative viability (%) = [(Asample - Ablank) / (Acontrol - Ablank)] × 100
Experimental controls
- Medium plus WST-8 reagent blank
- Untreated or vehicle-treated viable-cell control
- Positive cytotoxicity control when appropriate
- Compound-only background control for colored or reducing test substances
Important precautions
- Compounds or culture conditions that alter dehydrogenase activity may change the WST-8 signal independently of the actual number of viable cells.
- Reducing agents can directly convert WST-8 to formazan and increase background absorbance. Include compound-only controls when such interference is possible.
- Avoid bubbles before plate reading because they can distort optical measurements.
- Optimize cell density and incubation time for each cell line. Leukocytes and other weakly reducing cells may require longer incubation or higher cell numbers.
- For highly turbid cell suspensions, measure a reference wavelength at 600 nm or higher and subtract it from the 450 nm reading.
- If sterile filtration is required, use a compatible 0.2 µm membrane and verify assay performance after filtration.
- Test compounds with strong intrinsic color or absorbance near 450 nm require additional background controls.
- Use aseptic technique and follow the safety information supplied with the product.
Storage and handling
- Store refrigerated at approximately 4°C.
- Protect from prolonged light exposure during storage.
- Do not use beyond the expiry date stated on the AffiGEN product label or lot-specific documentation.
- A shelf life of up to 24 months may apply only when supported by the labeled expiry and verified storage conditions for the supplied lot.
Frequently asked questions
What does the assay measure?
The assay measures the reduction of WST-8 by metabolically active cells. The resulting 450 nm absorbance is used as an indicator of viable cell number within an optimized linear range.
Does the formazan product require solubilization?
No separate solubilization step is normally required because the WST-8 formazan product is water-soluble in the culture medium.
How long should the plate be incubated after adding the reagent?
A 1-4 hour incubation is a suitable starting range. The optimal time depends on the cell type, cell number and experimental treatment.
Can reducing compounds interfere with the assay?
Yes. Reducing agents may generate formazan independently of viable cells. Include test-compound background controls and subtract any non-cellular signal.
Why is my absorbance signal unexpectedly low?
Possible causes include low cell density, insufficient color-development time, weak metabolic activity, inappropriate culture conditions or treatment-induced metabolic suppression. Run a cell-density and incubation-time optimization experiment.
Ordering information
Product: AffiASSAY® WST-8 Cell Viability, Proliferation & Cytotoxicity Assay Kit (CCK-8 Type)
| Size | Estimated tests* | SKU |
|---|---|---|
| 1 mL | 100 Tests | AFG-MCX-1783-01 |
| 5 mL | 500 Tests | AFG-MCX-1783-02 |
| 6 × 5 mL | 3,000 Tests | AFG-MCX-1783-03 |
| 24 × 5 mL | 12,000 Tests | AFG-MCX-1783-04 |
*Test counts are based on using 10 µL of reagent per well.