**4. Discussion and conclusion**

Evaluating cellular cytotoxicity is one of the most essential parts of studying cellular functions in biology. These assays are used to test a substance's effects on *in vitro* systems in the fields of including but not limited to oncology, biotechnology, drug discovery, pharmacology, product development, and medical device biocompatibility testing [17, 22]. When exposed to cytotoxic compounds, cells can undergo necrosis, apoptosis, and autophagy, or they could stop proliferating. Detecting this dynamic event is crucial for evaluating the mechanisms in action of cellular actions and pathways involved in cell death after exposure to toxic agents.

There are a variety of assays that can be used in general, such as dye exclusion assays, colorimetric, fluorometric, and luminometric assays [23]. Here, in this chapter, we reported the uses of colorimetric cytotoxicity assays where data are recorded using a multiplate reader. The idea for all is to use a compound to treat the cell and addition of a dye that changes its absorption spectra upon cellular reduction, which is directly proportional to the number of metabolically active cells. Thus, the principles of cytotoxicity assays are different from cellular viability assays, which typically measure viable cells rather than the metabolic activity. As cytotoxicity and viability assays can be utilized separately, additional tests may also be required based on the research aim. As such, they could be used together as complementary methods to get a better understanding of a cell's metabolic reaction.

It should be noted that most of the cytotoxic measurement assays actively affect cellular integrity, protein production, cellular trafficking and alter the cell fate by activating programmed cell death [24]. This makes them irreversible assays where the cells could not be used after the assay. Nonetheless, they provide rapid, robust, sensitive, and cost-effective means to determine whether a material contains potentially biologically harmful activity or substances.

One of the key factors in selecting the assay/dye type in cytotoxicity assays is the biological endpoint. As some experiments require certain types of cells, cellular sources should be carefully selected depending on the endpoint used in the cytotoxicity test.


#### **Table 1.**

*Summary, advantages, and disadvantages of different colorimetric biological assays used in vitro.*

For example, in cytokine release studies, monocytes and fibroblasts should be the cell of choice [25, 26]. Similarly, cardiac-regeneration-related studies could be conducted with cardiac fibroblasts or cardiomyocytes for better results [27, 28].

Overall, colorimetric assays are simple, inexpensive, accurate, rapid, and sensitive methods in determining cellular toxicity. They are also applicable to both cell suspensions and adherent cells that make them attractive molecules in optimization studies. Although their main principle is based on metabolic activity determination, they all show some unique properties. We summarized their advantages and disadvantages in **Table 1**.

Main difference between the assays is that the MTT is not soluble in water, which requires an additional formazan dissolution step. In contrast, MTS, XTT, and WST assays use a different kind of tetrazolium salt, which produces a soluble formazan, reducing one step in the MTT assay procedure. Therefore, such assays are more efficient and less time-consuming when compared with the MTT assay.

It should be noted that environmental factors such as enzymatic regulation, pH, incubation time, temperature, cellular ion concentration, and variations in cell cycle could affect the performance of the colorimetric assays [29]. As such, an ideal cytotoxicity assay could differ from study to study depending on the aim, action mechanism, and environmental factors.
