**4.12 Trypan blue dye exclusion assay**

The trypan blue assay is a dye exclusion test that provides a straightforward and quick method for assessing cell viability. It is based on the principle that live cells possess intact cell membranes, which exclude the Trypan Blue dye, while dead or damaged cells take up the dye due to compromised membrane integrity. Viable cells have intact cell membranes, allowing them to exclude certain dyes (such as trypan blue, Eosin, and propidium), while dead cells cannot [45]. In this test, a cell suspension is mixed with trypan blue dye. The dye's absorption or exclusion is then visually examined, as viable cells will display clear cytoplasm, while nonviable cells will exhibit blue cytoplasm. A significant drawback of this technique is that it indirectly assesses viability based on cell membrane integrity. It is possible for a cell to be nonviable while still having an intact membrane. Conversely, cells with compromised membranes might recover and become fully viable. Another limitation is the subjective evaluation of dye uptake, which may cause small amounts of dye uptake to go undetected, potentially indicating cell damage.

One solution to this issue is to evaluate dye exclusion using a fluorescent dye and a fluorescence microscope instead of using trypan blue with a transmission microscope. However, determining dye uptake and cell viability using the cell's light scatter properties can be quite complex. A notable limitation of this method is that it is timeconsuming, although some protocols show that the trypan blue exclusion assay can be performed in under 10 minutes [46].

#### **4.13 GSH assay**

In human cells, the majority of glutathione (90–95%) is present in its reduced form (GSH). It plays a role in numerous regulatory processes, such as signal transduction, gene expression, DNA and protein synthesis, proteolysis, cell growth and apoptosis, cytokine and immune responses, protein glutathionylation, and the maintenance of mitochondrial function and integrity [47]. The glutathione assay is a colorimetric test that identifies alterations in GSH and GSSG levels during oxidative stress [48] using the enzymatic recycling technique with glutathione reductase and Ellman's reagent. This assay can measure reduced glutathione (GSH), oxidized glutathione (GSSG), total glutathione (GSH + GSSG) concentrations, and their ratio in various samples, including blood, plasma, serum, cultured cells, and tissues.

The glutathione reductase enzyme converts GSSG to GSH, generating a yellow chromophore that can be detected spectroscopically at 415 nm. Consequently, the concentration in an unknown sample is determined by evaluating the absorbance at 415 nm and comparing it to the standard curve for GSSG. This curve is plotted each time glutathione quantification is performed. The assay's sensitivity is enhanced by the enzyme glutathione reductase, which facilitates the enzymatic recycling of GSH. However, some drawbacks of the glutathione assay include its high polarity, limited stability, and the aliphatic structure of the assay [49].

#### **4.14 Protease viability marker assay**

The Protease Viability Marker Assay is a fluorescence-based method employed for assessing cell viability, cytotoxicity, and proliferation. This assay takes advantage of the presence of intracellular proteases, which are released from cells upon loss of membrane integrity, as a marker for cell viability [50]. These proteases specifically cleave nonfluorescent substrates, such as the commercially available Calcein-AM or CellEvent Caspase-3/7 Green Detection Reagent, to generate a highly fluorescent product, which can be detected using a fluorescence plate reader or a fluorescence microscope [32, 51].

One of the main advantages of the Protease Viability Marker Assay is its high sensitivity and specificity, as the fluorescent signal is only generated when the substrate is cleaved by the intracellular proteases, ensuring minimal background fluorescence. Additionally, the assay is nontoxic to the cells, allowing for real-time monitoring of cell viability over time and facilitating the assessment of cellular responses to various treatments or conditions [52]. However, there are some limitations to the Protease Viability Marker Assay. The assay may not be suitable for all cell types or conditions, as the presence and activity of intracellular proteases can vary depending on the cell type, culture conditions, or experimental treatments. Moreover, the fluorescent signal generated by the cleaved substrate may not be directly proportional to the number of viable cells, as the protease activity can be affected by various factors, such as cell density, cell size, or cell cycle stage. The Protease Viability Marker Assay can be combined with other assays to obtain more comprehensive information about cell

viability, cytotoxicity, and the mechanisms underlying cellular responses to various stimuli. For example, the assay can be used alongside assays that measure apoptosis, necrosis, or autophagy to provide a more complete picture of the cellular response to a test substance. By integrating the Protease Viability Marker Assay with complementary methods, researchers can gain deeper insights into the complex biological processes that govern cell survival and death in response to various stimuli.

### **4.15 Clonogenic cell survival assay**

The clonogenic cell survival assay, also known as colony formation assay, is a widely used method for evaluating the ability of cells to survive and proliferate following exposure to various stressors, such as radiation, chemotherapeutic agents, or other cytotoxic substances [53]. This assay is based on the principle that a single cell can give rise to a colony of cells, which can be counted and analyzed to determine the proportion of surviving cells with the ability to form colonies [53].

The assay involves seeding cells at a low density in culture dishes, followed by treatment with the agent of interest. After a suitable incubation period, typically 1–3 weeks, the cells are fixed, stained, and the number of colonies containing at least 50 cells is counted. The surviving fraction is calculated by comparing the colony formation efficiency of treated cells with that of untreated control cells. The clonogenic cell survival assay has several advantages. It provides a direct measure of the reproductive capacity of cells, allowing for the assessment of treatment-induced cytotoxicity at the level of individual cells. Additionally, the assay is highly sensitive and can detect changes in cell survival across a wide range of treatment doses [54]. There are also some limitations to the clonogenic cell survival assay. The assay can be time-consuming and labor-intensive, as it requires a long incubation period for colony formation and manual counting of the colonies. Additionally, the assay may not be suitable for all cell types, particularly non-adherent or slow-growing cells, which may not form distinct colonies under the experimental conditions [55].

#### **4.16 DNA synthesis cell proliferation assays**

DNA synthesis cell proliferation assays are a group of methods used to assess cell proliferation by measuring the incorporation of nucleotide analogs into newly synthesized DNA during the S phase of the cell cycle. These assays are valuable for studying the effects of various stimuli, such as growth factors and cytotoxic agents, on cell growth and division. One commonly used DNA synthesis cell proliferation assay is the 5-bromo-2′-deoxyuridine (BrdU) assay. BrdU is a thymidine analog that gets incorporated into newly synthesized DNA during the S phase of the cell cycle. After incorporation, the BrdU-containing DNA can be detected using specific antibodies, allowing for the quantification of proliferating cells. The BrdU assay has been used in various applications, including drug screening and evaluation of the cytotoxic effects of anticancer agents [56]. Another DNA synthesis cell proliferation assay is the 3H-thymidine incorporation assay. This assay involves the incorporation of radioactive tritiated thymidine (3H-thymidine) into newly synthesized DNA. The amount of 3H-thymidine incorporated into the DNA can be quantified using a scintillation counter, providing a measure of cell proliferation. The 3H-thymidine incorporation assay has been widely used in studies investigating cell proliferation in response to growth factors, cytokines, and other signaling molecules [57]. DNA synthesis cell proliferation assays offer several advantages. They provide a direct measurement of

DNA synthesis, reflecting cell proliferation rates, and they can be applied to a variety of cell types, including adherent and suspension cells. However, these assays also have some limitations, such as the potential for false-positive or false-negative results due to nonspecific incorporation of the nucleotide analogs and the need for proper controls to account for variations in DNA synthesis rates.

### **4.17 AGAR diffusion assay**

The agar diffusion test is a cytotoxicity barrier testing method in which the test material is simply placed on an agar layer covering a monolayer cell culture that simulates the mucosal membrane [58]. In this technique, cells are incubated for 24 hours before evaluating cytotoxicity. They are stained with neutral red dye to identify viable cells, stressed, and lysed [59]. Toxicity is determined by the loss of viable cells around the test material, which appears as an unstained area under and around the material being tested. In cases of high concentration and cytotoxicity of the diffusing substance, a loss of dye within the cells may be observed as the leachable toxic substance causes cell lysis [60]. Agar diffusion assays can evaluate the nonspecific cytotoxicity of the tested material's leachable components.

This test has the advantage of being cost-effective and simple to perform as a cytotoxicity screening tool. However, one limitation of this test is that potentially cytotoxic leachates in a solid state may stick to the agar rather than spreading across the plate, resulting in cells being only partially exposed to the test substance. Additionally, this test can only be used for materials that diffuse through the agar covering the cell monolayer. If materials do not dissolve in or spread through the agar, they will not show toxicity by damaging cells. Nonetheless, such materials could still be cytotoxic in a clinical setting [58].

#### **4.18 Raman micro-spectroscopy**

Raman micro-spectroscopy is a nondestructive and label-free optical technique that combines Raman spectroscopy with microscopy to provide detailed information about the molecular composition, structure, and interactions within a sample. This technique relies on the inelastic scattering of light, also known as Raman scattering, which occurs when light interacts with molecular vibrations in a sample [61]. Raman micro-spectroscopy has been widely used in various fields, including materials science, biology, and medicine. In materials science, it is used to study the crystallographic structures, stress distributions, and phase transitions of materials [62]. In biology and medicine, Raman micro-spectroscopy has been applied to study cellular processes, molecular interactions, and disease diagnosis.

One of the significant advantages of Raman micro-spectroscopy is its ability to obtain high-resolution spatial information about the sample's molecular composition without the need for labeling or sample preparation. This allows for real-time, in situ analysis of living cells, tissues, and biomaterials. Additionally, the technique is sensitive to both chemical and structural information, enabling the identification and differentiation of various molecular species within the sample [63]. However, Raman microspectroscopy also has some limitations. The most significant challenge is its inherently weak signal, which often requires long acquisition times or high laser power, which can cause sample damage or photobleaching. Recent advancements in instrumentation, such as the development of near-infrared lasers and highly sensitive detectors, have significantly improved the signal-to-noise ratio and reduced the acquisition time.
