Cell-viability assays are a cornerstone of biological research, offering crucial insights into cellular health and the effects of various stimuli on cellular systems, such as drugs, toxins, growth factors, and environmental changes¹. By measuring key parameters such as metabolic activity, membrane integrity, enzyme activity, and ATP content, researchers can determine whether cells are alive, dead, or undergoing stress.

Accurate and reliable measurement of cell viability is essential in various fields. In drug discovery, it helps identify potential therapeutics, optimize drug concentrations, reduce toxicity, and enhance therapeutic efficacy. In toxicology, it is crucial for assessing the safety profiles of chemicals, environmental toxins, and nanomaterials. In cell biology, precise viability measurements are key to understanding fundamental cellular processes, including proliferation, differentiation, apoptosis, and necrosis.

The water-soluble tetrazolium salt 1 (WST-1; also known as 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt) assay is a common laboratory technique used for striking a balance between simplicity and precision in cell-viability testing. Its widespread adoption underscores its utility in advancing scientific knowledge across disciplines.

This article aims to provide a broad understanding of the WST-1 assay, covering its underlying principles, applications, methodology, and essential troubleshooting strategies, enabling both novice and experienced researchers to effectively harness WST-1 assays in their studies. The biochemical mechanisms underlying the WST-1 assay and its association with cell viability are presented in detail, alongside its effective application in a range of experimental contexts. Common technical challenges are discussed, and approaches for troubleshooting are provided to ensure accurate data analysis and support the derivation of meaningful and reliable conclusions.

Unveiling the mechanism: The principle underlying the WST-1 assay

The WST-1 assay is a colorimetric assay that quantitatively assesses cell viability by measuring cellular metabolic activity. The assay is fundamentally based on the activity of mitochondrial dehydrogenases, which are the enzymes present in metabolically active cells. These enzymes catalyze the biochemical process of cleaving WST-1, resulting in the formation of a water-soluble formazan dye ². A microplate reader is used to quantify the amount of formazan dye produced by measuring absorbance in the UV-visible range³. The amount of formazan dye produced is directly proportional to the number of viable cells in the sample.

The biochemical reaction can be summarized as follows:

WST-1 (Tetrazolium Salt) + Mitochondrial Dehydrogenases + Electron Carriers → Formazan Dye (Soluble) + Oxidized Substrates

The reaction involves the transfer of electrons from NADH  or FADH₂, generated by mitochondrial dehydrogenases, to WST-1, resulting in WST-1’s reduction to the colored formazan product⁴.

WST-1, which is a negatively charged molecule that does not readily cross the cell membrane, undergoes reduction in the extracellular space, facilitated by electron-coupling reagents, such as 1-methoxy phenazine methosulfate, which mediate the shuttling of electrons from the mitochondrial electron transport chain across the cell membrane, enabling the formation of formazan⁵. The extracellular reduction mechanism prevents interference from intracellular processes, ensuring accurate measurements of cell viability.

Succinate-tetrazolium reductase, also known as succinate dehydrogenase, is a specific mitochondrial enzyme that oxidizes succinate to fumarate in the Krebs cycle. It transfers electrons to ubiquinone and subsequently to the electron transport chain6. The electrons then reduce the tetrazolium salt, leading to the formation of a colored formazan product. This activity of succinate-tetrazolium makes it a key player in the reduction of WST-1.

This spectrophotometric approach allows rapid and sensitive detection, making it ideal for drug screening, toxicity testing, and cell proliferation assays.

Versatility of the WST-1 assay

The WST-1 assay is a versatile tool applicable in several areas of cell biology. Its applications can be broadly categorized into assessments of cell proliferation and cytotoxicity, as well as drug sensitivity testing.

Cell proliferation and cytotoxicity assessments

The WST-1 assay enables the measurement of metabolic activity in cell populations over time and under varying growth conditions. This allows researchers to investigate the effects of growth factors, cytokines, and nutrients on cell proliferation, as exemplified by studies on T cell proliferation in response to interleukin-2 (IL-2)⁷.

The assay is also valuable for cytotoxicity assessments, allowing the evaluation of the impact of various substances on cells. A reduction in metabolic activity serves as a key indicator of cytotoxicity, as demonstrated by measuring the effects of tumor necrosis factor-alpha (TNF-α) on fibrosarcoma cell lines ⁸.

Drug-sensitivity testing

The WST-1 assay is crucial in evaluating the effectiveness of drugs, particularly anticancer agents such as cisplatin, triazole, and doxorubicin⁹. It allows for the determination of dose-response relationships between drug concentration and cell viability or cytotoxicity.

Cells are exposed to various drug concentrations, and the corresponding absorbance values are measured. Dose-response curves are then constructed, allowing for the calculation of key parameters, such as IC₅₀ and EC₅₀ values, which offer valuable insights for optimizing drug dosages and predicting treatment outcomes.

The suitability of WST-1 assays for high-throughput screening also makes it an excellent tool for rapid, automated screening of large compound libraries¹⁰. It can be used to screen thousands of compounds for their ability to inhibit cancer cell growth or to enhance the effectiveness of existing therapies. This approach can accelerate the discovery of novel drugs and improve treatment outcomes for cancer patients.

WST-1 vs. the rest: Advantages and disadvantages

Advantages of the WST-1 assay

• Non-radioactive method: Compared with radioactive assays, the WST-1 assay enhances laboratory safety by eliminating the risks associated with radioactive materials and simplifies waste disposal, thereby reducing regulatory and environmental concerns^11,12.
• Rapid results: WST-1 assay provides results between 30 minutes and a few hours, depending on experimental conditions and cell types. This rapid turnaround time is beneficial for high-throughput screening and time-sensitive experiments.
• Higher sensitivity: More sensitive than other tetrazolium-based assays such as MTT, XTT, and MTS, WST-1 can detect minute changes in cell viability and proliferation, making it suitable for experiments requiring precise measurements^13–16.
• One-step procedure: The reagent is added directly to the cell culture, eliminating the need for washing, harvesting, and solubilization steps. This streamlined protocol is easy to use, saves time, and reduces the risk of errors or cell damage during processing¹⁷.
• Water-soluble formazan product: Eliminates the need for organic solvents, such as DMSO, which are required in the MTT assay to solubilize formazan crystals.
• Time-course studies: Allow for multiple readings from the same well at different time points without disrupting the cells, providing insights into the dynamics of cellular responses and enhancing the assay’s versatility⁵.

Disadvantages and considerations of the WST-1 assay

• Intermediate electron acceptor requirement: Some formulations require an intermediate electron acceptor, which may pose potential toxicity to cells or interfere with assay results, therefore necessitating careful evaluation¹⁸.
• Higher background absorbance: Background absorbance may be higher in WST-1 assays than that in MTT assays, depending on the culture medium and pH¹⁶. This can reduce the assay’s sensitivity, requiring careful optimization of assay conditions.
• Interference from certain compounds: Manganese-containing materials may interfere with WST-1 reduction, affecting the results¹⁹. Likewise, compounds with antioxidant activity may influence assay outcomes by scavenging free radicals involved in the reduction process.
• Optimization for specific cell lines: Optimal conditions, such as seeding density and incubation time, may need to be determined beforehand for different cell lines. This can be time-consuming, but it ensures accurate and reliable results.
• Cost considerations: The reagents for the WST-1 assay may be more expensive than those for other one-step assays such as MTS²⁰. Researchers should weigh the cost against the assay’s advantages, such as higher sensitivity and ease of use.

Key comparison: WST-1 vs. MTT vs. MTS assays

A practical guide: Step-by-step WST-1 assay protocol

This section offers a practical guide to performing the WST-1 assay, detailing the necessary reagents, equipment, and a step-by-step procedure for reliable results.

Reagents and materials

Essential reagents include an appropriate cell-culture medium for the cell line being used. Fetal bovine serum (FBS) is needed to supplement the cell culture as required¹⁸. The WST-1 assay reagent should be a ready-to-use solution or components from a kit. Optional compounds are needed for testing proliferation, cytotoxicity, and drug sensitivity. Sodium dodecyl sulfate (SDS; 1%) as a stopping solution is also optional. Phosphate-buffered saline (PBS) is used for washing cells if needed. (Check out Abcam’s WST-1 assay reagent here.)

Equipment

Essential equipment includes 96-well flat-bottom tissue culture plates, a cell culture incubator maintained at 37°C with 5% CO₂ and appropriate humidity, and a microplate reader capable of measuring absorbance in the 420–480 nm range, with optimal absorption occurring at 440–450 nm, with a reference wavelength ideally above 600 nm for background-absorbance correction^13,18. Pipettes and sterile pipette tips are required, and a plate shaker for mixing is optional.

Procedure

To perform the WST-1 assay, follow these steps:

1.     Cell seeding: Seed cells into the wells of a 96-well plate at an optimized density for the specific cell line and experimental needs. The optimal density should be determined empirically for each cell type through cell-titration experiments.

2.     Incubation: Incubate the cells under standard cell culture conditions, typically for 24–96 h, depending on the experimental design.

3.     Addition of WST-1 reagent: Add WST-1 reagent directly to each well. A volume of 10 µL per 100 µL of culture medium is recommended; however, it is also advisable to follow the manufacturer instructions for specific guidelines and optimal assay performance¹³.

4.     Control wells setup:

• Blank control wells: Contain only culture medium and WST-1 reagent (no cells).
• Untreated control wells: Contain cells and culture medium without any test compounds (for proliferation assays) or with vehicle control (for drug testing).
• Positive and negative control wells: Include appropriate controls based on the experiment (eg, cells treated with a known cytotoxic agent or growth-promoting factor).

5.     Incubation with WST-1: Incubate the plate under standard conditions for an additional period, typically 0.5–4 hours¹³. The optimal incubation time should be determined for each experiment and cell type. Monitor color development to identify the ideal endpoint.

6.     Shaking the plate: Gently shake the microplate for 1 minute to ensure uniform distribution of the formazan dye in the wells.

7.     Absorbance measurement: Measure the absorbance of each well using a microplate reader. Absorbance should be measured at a wavelength between 420 and 480 nm, with maximum absorbance typically around 440 nm¹³. If available, use a reference wavelength above 600 nm to correct for background absorbance.

8.     Stopping the reaction (optional): If immediate reading is not possible, the reaction can be stopped by adding 10 µL of 1% SDS to each well²¹.

Navigating challenges: Troubleshooting tips and considerations

The WST-1 assay, while robust, can present challenges that may affect the accuracy and reliability of results. Even with a well-designed protocol, challenges may arise during the assay. This section outlines common issues, their potential causes, and solutions to ensure accurate and reliable results²².

Low absorbance readings

Low absorbance readings often indicate insufficient signal generation due to improper assay conditions.

Cell density significantly impacts the assay²³. Too few cells may result in signals below the detection limit. Too many cells, on the other hand, can cause signal saturation, leading to inaccurate results. Optimizing the initial cell-seeding density for each cell line is crucial to ensure measurable and reliable results.

Insufficient incubation time with the WST-1 reagent directly influences the amount of formazan produced and, therefore, the assay’s accuracy. Empirical determination of the duration of optimal incubation for different cell types and experimental conditions is essential for achieving accurate measurements.

Improper storage of the WST-1 reagent can cause degradation of the reagent, affecting the assay’s performance. Storing the WST-1 reagent properly at -20°C to prevent degradation and avoiding repeated freeze-thaw cycles are essential for maintaining reagent efficacy.

Incorrect microplate reader settings outside the 420–480 nm range or unhealthy cells with low metabolic activity may also contribute to low absorbance readings. To address this, confirm the microplate-reader settings and check cell health before starting the assay.

High background absorbance

Elevated background absorbance can interfere with accurate measurements. This may result from excessively long incubation times, interference due to culture-medium components such as phenol red or high FBS concentrations, contamination within wells, and premature exposure of the WST-1 reagent to light prior to use.

To minimize background absorbance, limit incubation time with WST-1, use blank controls to account for background signals, and maintain clean techniques throughout the experiment. Prolonged exposure of WST-1 to light can also increase background absorbance, affecting the accuracy of the assay; therefore, minimizing light exposure before and during the assay helps maintain the integrity of the measurements.

Inconsistent or variable results

Variability in the assay results can stem from uneven cell seeding (eg, clumped cells), temperature fluctuations during incubation, edge effects in multi-well plates, and insufficient mixing following WST-1 addition. To ensure consistency, a uniform single-cell suspension should be prepared for seeding.

Plates should be properly equilibrated to maintain stable conditions, including a temperature of 37°C and 5% CO₂, throughout both cell-culture and assay incubation. Ensuring proper and consistent environmental conditions during the assay supports cell health and accurate measurements. Furthermore, outer wells should be reserved for control conditions rather than experimental use. The multi-well plate should be gently shaken to ensure uniform distribution of the formazan dye within the wells, thereby promoting consistent and reliable color development.

Interference from test compounds

Certain test compounds may interfere with the WST-1 assay by non-enzymatically reducing the tetrazolium salt or interacting with assay components. For example, manganese-containing materials or antioxidant compounds may alter results¹⁹. To detect potential interference, control wells containing only the test compound and WST-1, but no cells, should be included in the experimental setup. If interference is confirmed, consider using an alternative viability assay to validate findings.

General tips for successful assay performance

To maximize reliability and accuracy, ensure all reagents are at the appropriate temperature before use and avoid introducing air bubbles during handling. Use fresh reagents whenever possible. Perform all measurements in duplicate or triplicate to account for variability and apply statistical analysis methods such as t-tests or ANOVA to confirm significance in results^24,25.

Sterile techniques

Using sterile techniques throughout the procedure is critical. This helps prevent contamination of cell cultures and reagents, ensuring reproducibility.

Calibration and functioning of equipment

Ensuring that the microplate reader is properly calibrated and functioning correctly is essential for guaranteeing accurate absorbance measurements. This includes regular checks and maintenance according to the manufacturer's guidelines.

Interpreting and analyzing WST-1 data

Background subtraction

Background subtraction is performed to account for absorbance arising from the culture medium or the WST-1 reagent itself, thereby ensuring accurate quantification of the cell-derived signal. The absorbance of blank control wells containing only culture medium and the WST-1 reagent should be measured across multiple wells, and the average of these values should be subtracted from all other wells.

Cell viability calculation (cytotoxicity assays)¹⁶

The formula for cell viability:

Cell viability is typically expressed as a percentage of the control (untreated) cells. The formula for calculating cell viability is:

Cytotoxicity calculation:

Cytotoxicity is the inverse of cell viability and represents the percentage of cells that have been killed or damaged by a treatment. It can be calculated using either of the following formulas:

Drug sensitivity testing and dose-response curves

Drug sensitivity testing involves generating dose-response curves to assess drug effectiveness²⁶. Plot the percentage of cell viability (or cytotoxicity) on the Y-axis against drug concentration (typically on a logarithmic scale) on the X-axis. From this curve, determine the IC₅₀ value, which indicates the drug concentration required to inhibit 50% of cell viability²⁷.

Generating a standard curve (for absolute cell viability)

To determine the absolute number of viable cells, generate a standard curve. Plate wells with a known number of cells, perform the WST-1 assay, and plot absorbance values against the corresponding cell numbers. Use this standard curve to extrapolate the number of viable cells in the unknown samples based on their absorbance readings.

Statistical analysis

Appropriate statistical analysis is essential for interpreting results accurately. If the data are normally distributed, use t-tests or ANOVA to compare different treatment groups or conditions^24,25, otherwise, a Mann-Whitney U test or a Kruskal-Wallis test can be used. A minimum of three replicates should be included for each experimental condition to ensure the generation of reliable and statistically meaningful results. Present data using graphs and tables, including error bars that represent the standard deviation or standard error of the mean to illustrate data variability. Report p-values to indicate the statistical significance of differences between groups. A p-value ≤ 0.05 is typically considered statistically significant²⁸.

Adherence to these guidelines for data interpretation and analysis enables the extraction of meaningful insights from WST-1 assay results, thereby supporting informed conclusions and contributing to a deeper understanding of cellular processes.

Maximizing accuracy: Best practices for optimizing WST-1 assays

To ensure the WST-1 assay yields accurate and reproducible results, consider the following best practices across various aspects of the experimental design and execution.

Consistent cell culture conditions: Maintaining consistent cell culture conditions minimizes variability. This includes:

• Use of same passage number: Ensure all cells used are from the same passage number.
• Consistency in growth medium and serum: Use the same batch of growth medium and serum throughout the experiment.

Including appropriate controls is crucial for accurate data interpretation.

• Blank control: Include a blank control (culture medium + WST-1 reagent) to measure background absorbance.
• Untreated control: Include untreated cells in the culture medium to serve as the baseline for cell viability.
• Positive/negative control: Use positive (known cytotoxic agent) and negative (growth-promoting factor) controls to ensure assay reliability.

Optimizing WST-1 assays requires careful control of various factors, such as cell density, incubation time, temperature, CO₂ levels, light exposure, proper shaking of the microplate, and reagent storage, along with consistent cell culture conditions and appropriate controls to ensure accuracy and reproducibility of results.

Conclusion

The WST-1 assay has emerged as a vital and widely adopted technique in the field of cell biology, providing a reliable, sensitive, and user-friendly approach to evaluating cell viability, proliferation, and cytotoxicity. This article has delved into every critical aspect of the assay, from the fundamental principles that govern its colorimetric detection mechanism to the detailed step-by-step protocols for optimal execution. Furthermore, it has provided practical tips for troubleshooting and offered insights into interpreting results accurately.

The versatility of the WST-1 assay makes it an indispensable tool in various research domains, including pharmacology, toxicology, cancer biology, and regenerative medicine. More importantly, its water-soluble nature allows for continuous monitoring of live cells, minimizing experimental disruption and ensuring reproducibility.

However, the accuracy and reliability of results are significantly influenced by factors such as cell type, incubation time, and reagent handling. By carefully designing experiments, researchers can leverage the full potential of the WST-1 assay to produce high-quality, reproducible data that drives scientific progress. Ultimately, the use of this technique is critical to expanding our understanding of cellular mechanisms and accelerating the development of therapeutic strategies.

FAQs

1. What types of cells can be used with the WST-1 assay (adherent or suspension cells)?

Both adherent and suspension cells can be used with the WST-1 assay. However, optimization of cell seeding density and assay conditions may be necessary for different cell types.

2. Can WST-1 assays be performed simultaneously with other assays such as ELISA or BrdU?

The WST-1 assay is typically performed as a standalone assay due to the need for specific incubation and reading conditions. Although not typically performed simultaneously, WST-1 can be used in conjunction with other assays on the same cells sequentially without mutual interference, which may increase the efficacy and consistency of these measurements to a certain extent.

3. Why might WST-1 results differ from other assays like ATP-based luminescence assays?

WST-1 measures mitochondrial dehydrogenase activity, while ATP assays measure ATP levels. These reflect different aspects of cell viability and metabolism. Differences may arise owing to variations in metabolic state or specific cellular stress responses that affect mitochondrial activity and ATP production differently.

4. How do I choose the optimal seeding density for my cells in the WST-1 assay?

Optimal seeding density is cell-type specific and should be determined empirically. A cell titration experiment should be performed by seeding cells across a range of densities (eg, 1,000–50,000 cells per well in a 96-well plate), followed by the WST-1 assay. The optimal cell density should then be selected based on the generation of a robust signal that remains within the assay’s linear detection range.

5. What are the key considerations for selecting the appropriate WST-1 reagent and electron coupling reagents?

Consider the specific requirements of your cell type and experimental conditions. Some WST-1 formulations may be more suitable for certain cell types or have different sensitivities. Electron coupling reagents should be carefully selected to ensure they are non-toxic to the cells and do not interfere with the assay readout. Always adhere to the manufacturer recommendations for reagent selection and use.

6. How can I minimize edge effects in WST-1 assays performed in 96-well plates?

Edge effect, where outer wells exhibit different results due to temperature variations or evaporation, is a common problem in 96-well formats, leading to variances between readouts of the outer wells compared with the central wells of a multi-well plate. It can be minimized by using the outer wells only for blank controls, ensuring consistent incubator temperature and humidity, reducing assay time, and using plate sealers or humidified incubators to reduce evaporation.

7. What is the typical linear range of the WST-1 assay, and how can I ensure my results fall within this range?

The linear range of the WST-1 assay depends on the specific cell type, reagent, and microplate reader. To determine the linear range, perform a cell titration experiment and plot absorbance values against cell numbers. Ensure that the cell densities used in your experiments fall within the linear portion of this curve.

References

1. Riss, T. L., Moravec, R. A., Niles, A. L., et al. Cell viability assays. Assay guidance manual [Internet] (2016).

2. Nemudzivhadi, V., & Masoko, P. In vitro assessment of cytotoxicity, antioxidant, and anti‐inflammatory activities of Ricinus communis (Euphorbiaceae) leaf extracts. Evidence‐based Complementary and Alternative Medicine, 2014, 625961 (2014).

3. Kirkland, D., Brock, T., Haddouk, H., et al. New investigations into the genotoxicity of cobalt compounds and their impact on overall assessment of genotoxic risk. Regulatory Toxicology and Pharmacology, 73, 311-338 (2015).

4. Berridge, M. V., & Tan, A. S. Trans-plasma membrane electron transport: a cellular assay for NADH-and NADPH-oxidase based on extracellular, superoxide-mediated reduction of the sulfonated tetrazolium salt WST-1. Protoplasma, 205, 74-82 (1998).

5. Sarı, C., Kolaylı, S., & Eyüpoğlu, F. C. A comparative study of MTT and WST-1 assays in cytotoxicity analysis. Haydarpaşa Numune Medical Journal, 61, 281 (2021).

6. Rutter, J., Winge, D. R., & Schiffman, J. D. Succinate dehydrogenase–assembly, regulation and role in human disease. Mitochondrion, 10, 393-401 (2010).

7. Alam, S., Katakura, Y., Yoshida, H., et al. Functional characterization of human T cells immortalized by oncogene transfection. Cytotechnology, 23, 185-192 (1997).

8. Fushimi, K., Torigoe, K., Yamauchi, H., et al. Establishment of a human fibroblast cell line producing tumor necrosis factor α (KMST-6/TNF) and growth inhibitory effects of its conditioned medium on malignant cells in culture. In Vitro Cellular & Developmental Biology-Animal, 34, 463-467 (1998).

9. Bębenek, E., Kadela-Tomanek, M., Chrobak, E., et al. Novel triazoles of 3-acetylbetulin and betulone as anticancer agents. Medicinal Chemistry Research, 27, 2051-2061 (2018).

10. Johnson, P. H., Walker, R. P., Jones, S. W., et al. Multiplex gene expression analysis for high-throughput drug discovery: screening and analysis of compounds affecting genes overexpressed in cancer cells. Molecular cancer therapeutics, 1, 1293-1304 (2002).

11. Elgqvist, J., Timmermand, O. V., Larsson, E., et al. Radiosensitivity of prostate cancer cell lines for irradiation from beta particle-emitting radionuclide 177Lu compared to alpha particles and gamma rays. Anticancer research, 36, 103-109 (2016).

12. National Academies of Sciences, Engineering, and Medicine. Radioactive Source Uses, Risks, and Control. In Radioactive Sources: Applications and Alternative Technologies. National Academies Press (US) (2021).

13. Ngamwongsatit, P., Banada, P. P., Panbangred, W., et al. WST-1-based cell cytotoxicity assay as a substitute for MTT-based assay for rapid detection of toxigenic Bacillus species using CHO cell line. Journal of microbiological methods, 73, 211-215 (2008).

14. Van Meerloo, J., Kaspers, G. J., & Cloos, J. Cell sensitivity assays: the MTT assay. Cancer cell culture: methods and protocols, 237-245 (2011).

15. Kuhn, D. M., Balkis, M., Chandra, J., et al. Uses and limitations of the XTT assay in studies of Candida growth and metabolism. Journal of clinical microbiology, 41, 506-508 (2003).

16. Riss, T. L., Moravec, R. A., Niles, A. L., et al. Cell viability assays. Assay guidance manual [Internet] (2016).

17. Gavanji, S., Bakhtari, A., Famurewa, A. C., et al. Cytotoxic activity of herbal medicines as assessed in vitro: a review. Chemistry & Biodiversity, 20, e202201098 (2023).

18. Yin, L. M., Wei, Y., Wang, Y., et al. Long term and standard incubations of WST-1 reagent reflect the same inhibitory trend of cell viability in rat airway smooth muscle cells. International Journal of Medical Sciences, 10, 68 (2012).

19. Scarcello, E., Lambremont, A., Vanbever, R., et al. Mind your assays: Misleading cytotoxicity with the WST-1 assay in the presence of manganese. PloS one, 15, e0231634 (2020).

20. López, I., Balderas-Rentería, I., Gómez, I., et al. MTT Vs WST-1, efficiency, cost, time, and waste generation: evaluating the silver nanoparticles cytotoxicity. Revista de Ciencias Farmacéuticas y Biomedicina, 3, 3-12 (2020).

21. Septisetyani, E. P., Ningrum, R. A., Romadhani, Y., et al. Optimization of sodium dodecyl sulphate as a formazan solvent and comparison of 3-(4,-5-dimethylthiazo-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay with wst-1 assay in mcf-7 cells. Indonesian Journal of Pharmacy, 25, 245 (2014).

22. Guadagnini, R., Halamoda Kenzaoui, B., Walker, L., et al. Toxicity screenings of nanomaterials: challenges due to interference with assay processes and components of classic in vitro tests. Nanotoxicology, 9(sup1), 13-24. (2015).

23. Gasque, K. C. D. S., Al-Ahj, L. P., Oliveira, R. C., et al. Cell density and solvent are critical parameters affecting formazan evaluation in MTT assay. Brazilian archives of Biology and Technology, 57, 381-385 (2014).

24. Wadhwa, R. R., & Marappa-Ganeshan, R. T test. In StatPearls [Internet]. StatPearls Publishing (2023).

25. Kim, T. K. Understanding one-way ANOVA using conceptual figures. Korean journal of anesthesiology, 70, 22 (2017).

26. Srinivasan, B., & Lloyd, M. D. Dose–Response Curves and the Determination of IC₅₀ and EC50 Values. Journal of Medicinal Chemistry, 67, 17931-17934 (2024).

27. Sebaugh, J. L. Guidelines for accurate EC₅₀/IC₅₀ estimation. Pharmaceutical statistics, 10, 128-134 (2011).

28. Tenny, S., & Abdelgawad, I. Statistical significance. In StatPearls [Internet]. StatPearls Publishing (2017).