Viability assay preference mostly depend on our requirement and the type of cells used. MTT is one of the most common viability assay that can be used for adherent cultures. If we are interested in a suspension culture, we can use MTS as there are no washing steps involved in the procedure, unlike in MTT.

1. I prefer to use Alamar blue assay as the cells remain live and metabolically active following it, allowing me to use them for further Biochemical assays (ARS, ALP, etc.).

2. The benefit is the same: the cells remain alive, so two assays can be carried out within the same sample.

3. No, I have not found any challenges to this assay, the only limitation is that the chemicals are a bit expensive.

4. The best way to select a viability assay is to read the principles and mechanism of that assay in great detail and read the literature on the same topic as yours and the same cell type to check which assays have, they used for their viability analysis as invariably they must have gone through the same dilemma as yours.

A thorough information is required about the conditions of your cell culture and the metabolic activity of your cell to ensure the best compatibility between the viability assay and your cells.

CCK-8

Najah Elmounedi Is there a specific reason why you're using this assay?

Omama Syed Compared to MTT, in which the cell cannot survive after the reagent has been added, the cell survival rate for Cell Counting Kit-8 is over 90% even after 24 hours of incubation. Therefore, after the assay with Cell Counting Kit-8, those cells can be used for other experiments. However, it is necessary to wash the cells so that no dye remains on the cell surface.

I think that trypan blue dye exclusion is a very good viability test for cells in suspension, whereas for adherent cells measurement of LDH release is very efficient when you take into account the Total LDH content of untreated cells (taken as a control). I guess that MTT assay is not a good test of viability, since in a given living cell population, the reduction of the dye is not uniform and greatly varies from one cell to another (when loking under the light microscope).

Hello Omama,

which colouring makes the most sense also depends on the aim of the examination. We have favoured NR for simple toxicity tests. Firstly because of the uncomplicated handling and secondly because of the possibility of being able to follow up the photometric evaluation of the NR staining result with a KZ staining. This provides information about the protein mass of the cells and can provide additional information in relation to the NR staining.

Greetings

Jochem Spieker

*Most reliable assays:*

1. *ATP-based assays (e.g., CellTiter-Glo)*: Measure ATP levels, indicating cellular energy status. Reliable, sensitive, and accurate.

2. *Resazurin-based assays (e.g., Alamar Blue)*: Detect cellular metabolism and reduction potential. Simple, non-toxic, and versatile.

3. *Trypan Blue exclusion assay*: Measures membrane integrity, distinguishing live and dead cells. Inexpensive, simple, and widely used.

*Features and benefits:*

- *Sensitivity and accuracy*: ATP-based assays are generally more sensitive and accurate.

- *Non-toxicity*: Resazurin-based assays are non-toxic, allowing for repeated measurements.

- *Ease of use*: Trypan Blue exclusion assay is simple and requires minimal equipment.

- *High-throughput compatibility*: ATP-based and resazurin-based assays are adaptable to high-throughput screening.

*Challenges and limitations:*

- *Interference*: Fluorescent compounds or high background fluorescence can interfere with assays.

- *Cell type and density*: Assay performance may vary depending on cell type and density.

- *Toxicity*: Some assays (e.g., MTT) can be toxic to cells, limiting repeated measurements.

*Tips and recommendations:*

1. *Choose an assay compatible with your cell type and experimental conditions*.

2. *Optimize assay parameters* (e.g., incubation time, concentration) for your specific needs.

3. *Use multiple assays* to validate results, if possible.

4. *Consider non-toxic assays* for repeated measurements or long-term studies.

5. *Consult assay manufacturer guidelines* and scientific literature for best practices.

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The selection of a cell viability assay depends on various considerations, including the type of cells being studied, the experimental setup, and the level of accuracy required. Different assays offer distinct advantages and disadvantages, such as MTT assays' sensitivity but potential toxicity, ATP-based assays' high accuracy but need for specialized equipment, and resazurin-based assays' non-toxicity and simplicity but possible lower sensitivity. Trypan Blue exclusion assays are widely used and cost-effective but may lack accuracy, while Calcein AM assays provide high sensitivity and non-toxicity but may have increased background fluorescence. By weighing these factors and assay characteristics, researchers can choose the most appropriate method for their specific research needs, ensuring dependable and precise cell viability measurements.

The reliability of a cell viability assay depends on the specific context, such as the cell type, experimental goals, and conditions. However, one of the most widely used and reliable assays that I have used is the MTT assay. There is a reason why I consider it the most reliable one.

# MTT Assay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)

The MTT assay measures cell viability based on the activity of mitochondrial dehydrogenase enzymes, which reduce the MTT reagent (a yellow tetrazolium salt) into formazan crystals (purple). Only the metabolically active (viable) cells can do it. The intensity of the purple color is measured spectrophotometrically to correlate with the number of viable cells.

# Since it directly measures mitochondrial function, it reflects cellular metabolic activity, which is a good indicator of viability. The MTT assay has been extensively validated and widely used, making it a gold standard for many cell viability studies. There’s also a wealth of comparative data available. It provides reliable and quantitative data, and its reproducibility across different labs and cell types makes it a trusted choice.

*Alternative Assays

While the MTT assay is highly reliable, there are other popular assays that have their own advantages depending on the situation:

• Resazurin (Alamar Blue) Assay- Resazurin is reduced by viable cells into resorufin, a fluorescent compound. This assay is non-toxic and can be used for continuous monitoring. Advantages: Non-toxic and can be used for live-cell tracking. More sensitive than MTT in some cases, particularly for low cell numbers. Challenges: Resazurin can sometimes interfere with certain compounds or cell types, leading to variable results.
• ATP-Based Assay (e.g., CellTiter-Glo):How it Works: Measures cellular ATP levels, which is an indicator of viable, metabolically active cells. It uses luciferase to produce a luminescent signal proportional to ATP concentration. Advantages: Very sensitive and quick. It’s ideal for high-throughput screening. Challenges: ATP levels can fluctuate with factors other than viability (e.g., stress, metabolic rate), and ATP is rapidly depleted in dying cells, potentially giving an underestimated readout of viability.
• Trypan Blue Exclusion Assay: Trypan blue dye stains dead cells with compromised membranes, allowing for counting of viable (unstained) cells versus dead cells under a microscope. Advantages: Simple, direct, and can be used to distinguish live vs. dead cells visually. Challenges: Subjective and labor-intensive compared to colorimetric or luminescent assays. Also, it doesn't account for metabolic activity.
• LDH Release Assay: Measures lactate dehydrogenase (LDH), an enzyme released into the culture medium upon cell membrane damage or cell death. Advantages: Good for detecting cell death by necrosis or membrane damage. Challenges: LDH assays only measure dead or dying cells and are not useful for estimating the number of viable cells. Also, it can be affected by background LDH from serum.
• For a general-purpose, MTT remains a preferred choice because of its reproducibility, and quantitative measure of cell viability, though the choice of assay depends on experimental context.

BrdU Cell Proliferation Assay

MTT and Resazurin

The question posed and responses confuse proliferation with viability. Viability is associated with assessing the alive versus the dead of at least 100 individual cells. It is then reported as a percentage. For example, 77 out of 100 cells examined were alive and so had a 77% viability. Flow cytometry with LIVE/DEAD staining or apoptosis/dead staining (annexin V and PI) are classic approaches. As mentioned, trypan blue or similar dye exclusion approaches (eosin, congo red, or erythrosine B stain assays) have long been used, but are time consuming. However, PI, 7-AAD, and Calcein dyes can also be used to advantage.

Mtt

Please read "The MTT Assay: Utility, Limitations, Pitfalls, and Interpretation in Bulk and Single-Cell Analysis" doi: 10.3390/ijms222312827 I find the reported studies to be well executed. A few quotes from the manuscript include the following:

Moreover, the common purporting as a viability assay is often erroneous. Reduction of the dye depends primarily on cell metabolism; sometimes this is reflective of cell viability, but confounding variables means this often leads to the inaccurate utility of the assay.

To infer cell viability based on OD values, the assay may still suffer from unintended bias for further reasons.

Consequently, even under optimized conditions with stringent controls when the MTT assay is used to measure cell viability and/or metabolic activity, the results should be, where possible, confirmed with complementary assays to attain a more comprehensive perspective of the treatment response.

Complementary assays on cell viability or metabolic activity could also be considered to provide more robust data.

To summarize, as a tool to measure the cell viability, metabolic activity of cells, and/or treatment cytotoxicity, the MTT assay necessitates several considerations (Table 1). These may be addressed by performing extra optimization experiments which could be a time-consuming and tedious, yet important, process. In many cases, complementary assays are recommended to assist in the interpretation of MTT assay measurements. Nevertheless, the assay has been commonly used as the main basis of such measurements, overlooking the limitations and the necessity of performing optimization assays.

I recommend Resazurin/alamarBlue. The measurement is colorimetric. We measure the metabolism of Resazurin (the color of the medium to which Resazurin is added). After the test, the cells can be used in other tests.