Cytotoxicity assays for cancer drug screening: methodological insights and considerations for reliable assessment in drug discovery

Ensaios de citotoxicidade para triagem de drogas contra o câncer: insights metodológicos e considerações para avaliação confiável na descoberta de drogas

Abstract

The importance of cytotoxicity assays in in vitro drug discovery investigations has led to their rising profile. Drugs and other substances can disrupt cell membranes, limit protein synthesis, and bind irreversibly to receptors, all of which lead to cell death in cancer cells. To precisely measure the cell death resulting from these damages, one must choose a cytotoxicity test that meets specific criteria. A systematic search strategy was used to gather grey literature from 2001 to 2024, utilizing databases such as PubMed and Google Scholar. Specific keywords related to colorimetric, fluorometric, and dye exclusion assays, as well as “cytotoxicity,” were employed. Here, we only focus on screening drug cytotoxicity for cancer cells. This review discusses various cytotoxicity assays, such as “dye exclusion assays,” “colorimetric assays,” and “fluorometric assays.” It is crucial to prioritize safety, speed, reliability, efficiency, and cost-effectiveness, while also ensuring minimal interference with the test compound. Commonly used in toxicology and pharmacology, cytotoxicity assays are based on several biological processes. Selecting the correct assay method requires considerations such as assay specificity and sensitivity, detection mechanism, test drug properties, and laboratory availability. This review aims to assist researchers in performing reliable cytotoxicity assessments by providing insights into assay choices.

Keywords:
cytotoxicity assays; colorimetric assay; fluorometric assay; MTT; CFDA-AM; resazurin; MTS; XTT; CVS; WST-1; WST-8; SRB; in vitro assay

Resumo

A relevância dos ensaios de citotoxicidade em investigações in vitro para avaliação biológica tem levado ao seu perfil crescente em vários campos. Drogas e outras substâncias podem danificar as membranas celulares, limitar a síntese de proteínas e se ligar irreversivelmente aos receptores, resultando na morte de células. Para quantificar com precisão a morte celular causada por esses danos, é necessário selecionar um teste de citotoxicidade que atenda a requisitos específicos. Uma estratégia de pesquisa sistemática foi usada para reunir literatura cinzenta de 2001 a 2024, utilizando bancos de dados como o PubMed e o Google Scholar. Palavras-chave específicas relacionadas a ensaios colorimétricos, fluorométricos e de exclusão de cor, bem como “citotoxicidade”, foram empregadas. Aqui, focamos apenas em rastrear a citotoxicidade de drogas para células cancerígenas. Vários ensaios de citotoxicidade são discutidos nesta revisão, incluindo “ensaios de exclusão de corantes”, “ensaios colorimétricos” e “ensaios fluorométricos”. Há necessidade urgente de considerar a importância de colocar a segurança, a velocidade, a confiabilidade, a eficiência e a rentabilidade em primeiro lugar, ao mesmo tempo assegurando que o composto de teste é manuseado com interferência mínima. Comumente usados em toxicologia e farmacologia, os ensaios de citotoxicidade baseiam-se em uma série de processos biológicos. Selecionar o método de ensaio correto requer considerações como especificidade e sensibilidade do ensaio, mecanismo de detecção, propriedades do medicamento de teste e disponibilidade de laboratório. O objetivo desta revisão é ajudar os pesquisadores a realizar avaliações de citotoxicidade confiáveis, fornecendo insights sobre a escolha dos ensaios.

Palavras-chave:
ensaios de citotoxicidade; ensaio colorimétrico; ensaio fluorométrico; MTT; CFDA-AM; resazurina; MTS; XTT; CVS; WST-1; WST-8; SRB; ensaio in vitro

1. Introduction

Cell death plays a pivotal role in development and tissue homeostasis, ensuring the balance between cell proliferation and elimination in maintaining physiological processes. It encompasses both programmed cell death and necrosis and is fundamental in various cellular and developmental events, such as embryogenesis, where it removes unwanted cells. In therapeutic contexts, cell death eliminates overproliferated or faulty cells, aiding in the restoration of normal cell function and promoting overall health. Preclinical trials screen numerous natural products derived from plants and marine sources, along with synthetically modified compounds, before they advance to clinical phases in cancer drug discovery (Chaudhry et al., 2020, 2021c, 2022a, b; Jan & Chaudhry 2019c; Channar et al., 2023). The cultivation of cells is fundamental to the process of drug development since it forms the basis for the screening, identification and improvement of new medications. High-throughput screening methods in cytotoxicity research examine large libraries of molecules, natural extracts, or specimens for potential antitumor activity. To ascertain efficacious compounds, it is crucial to distinguish between live, dead, or disrupted cells. There are numerous techniques available for quantifying cell count and assessing cell viability (Sylvester, 2011). Assessing the number of live cells present in each sample determines cell viability. In any type of cell culture, the assessment of cell viability is critical. Often, the experiment's primary goal is to conduct cytotoxicity assays, but it also serves to establish a correlation between cell behavior and cell count (Stoddart, 2011). Primarily, we use cell viability assays to evaluate the cellular reaction to a pharmacological or chemical agent. The pharmaceutical industry extensively employs viability assays to assess the impact of newly produced drugs on cells. Scientists use various assays to evaluate the efficiency of developing drugs, which often focus on targeting cancer cells (Adan et al., 2016). Cytotoxicity describes the hazardous effects of a molecule or substance on cells. The field of study is crucial for assessing the degree of toxicity that a molecule or chemical substance might inflict on cells (Vinken and Blaauboer, 2017). Cytotoxicity tests employ various cellular processes. These include “cell membrane permeability,” “enzymatic activity,” “cell adhesion,” making ATP, coenzymes, and nucleotides (Ishiyama et al., 1996). At the end of the experiment, it is important to determine the number of living cells and the number of dead cells. Toxicology and pharmacology currently employ various cytotoxicity or viability assays. The test method selection is critical in determining the nature of the interaction (Aslanturk, 2018). These tests usually check for dead cells in two main ways: (a) using a dye that can get into cells with damaged membranes, and (b) measuring marker molecules that can only get into compartments when the cell membrane is damaged. So, these cytotoxicity tests are very helpful for learning about many aspects of cell health, such as cell membrane integrity, metabolism, cellular machinery, proliferation, and programmed cell death (Maier et al., 1991; Fotakis and Timbrell, 2006; Yunus et al., 2020; Gul I et al., 2021; Yunus et al., 2024). Many of these assays, recognized for their sensitivity and reproducibility, employ colorimetric and fluorometric techniques, necessitating the utilization of diverse dyes (Ude et al., 2022). This review is dedicated to examining in vitro approaches for cytotoxicity analysis, descriptive methodology, and proper criteria for selection.

2. Review Methodology

A comprehensive search was performed in credible publishing databases like PubMed and Google Scholar to gather grey literature published from January 2001 to August 2024. utilized a search technique that required article titles to include terms such as “colorimetric assay,” “fluorometric assay,” and “dye exclusion assay,” along with an extra keyword that indicated the specific term associated with the assays, like “cytotoxicity.” The title and abstract were examined to assess their initial eligibility. We conducted an evaluation to determine the appropriateness of the entire manuscript. Academic papers with limited access were obtained from Universiti Terengganu Malaysia. Abstracts from the chosen papers were examined after an initial assessment of their titles. After conducting a thorough evaluation of each article using specific criteria for inclusion and exclusion, we carefully selected the articles that would be included in the study. A wide range of sources were utilized to gather grey literature, including search databases that covered books, dissertations, working papers, and government publications. At present, exclusion restrictions only apply to languages other than English.

2.1. Significance and applicational considerations for cytotoxicity assays

According to Eisenbrand et al. (2002), cytotoxicity is primarily defined as the ability of a chemical to cause changes in cellular behavior and processes that ultimately lead to cells dying. In other words, cytotoxicity in cells is defined as the concentration at which a test material or substance can cause 50% of the cell to die. The inhibitory concentration, also known as IC50, quantifies the expression by calculating the average percentage increase compared to the untreated control group (He et al., 2016; Shafiee et al., 2021). Similarly, an alternative method in the toxicological sciences is the cytotoxicity test, which involves an in vitro study to measure various parameters related to cell death and proliferation (Mahto et al., 2010). Cytotoxicity assays play a vital role in various fields such as basic research, materials science, ecological evaluation, and the pharmaceutical sector. They are particularly significant in the development of anticancer therapies, as they help researchers gain insights into the susceptibility of cancer cells to new treatments. In natural products, which may include marine, plant-based lead compounds, and various metal-based synthetic complexes, screening analysis is a major initiation in the path towards the discovery of new drugs. Measuring the enzymatic conversion of color compounds (calorimetric, florescence, dye) in living cells directly highlights the percentage of cell growth inhibition (Yunus et al., 2024; Chaudhry et al., 2024a, 2024b). Screening natural products is seen as an important part of both in vitro and in vivo biomarker analysis when it comes to natural product dosage and safety (Chaudhry et al., 2024c). The research community commonly employs fluorescent and colorimetric assays for cytotoxicity assessment due to their significant popularity (Stoddart, 2011; Garcia-Hernando et al., 2020). However, the determination of whether research is assessing the capacity of substances to inhibit or promote cell proliferation and migration, or trigger cell death is entirely contingent upon the specific goal of an investigation. There are also animal models that can be used for cytotoxicity tests, but in vitro cell culture has always been the best way to see how biological materials or active chemicals affect cells (Kunzmann et al., 2011). The assay's specificity and sensitivity, the detecting mechanism, the test drug's characteristics, and the availability of the laboratory are all crucial factors to consider when choosing the right assay method. Figures 1 and 2 categorize the assays as follows: i) colorimetric assays; ii) dye exclusion assays; and iii) fluorometric assays.

Figure 1
Major types of cytotoxicity detection methods.

Figure 2
A visual representation of A) Colorimetric, B) Dye exclusion and C) Fluorometric detection assays.

2.2. Colorimetric assays

Colorimetric methods rely on tetrazolium salts to induce color changes. In 1983, Mossman introduced the first colorimetric approach as a substitute for the laborious, time-consuming, and expensive radioactive techniques used to estimate the viability and growth of mammalian cells (Mosmann, 1983; Karatop et al., 2022). The key idea behind these tests is to measure a biochemical marker to assess cells' metabolic activity. We typically measure common reducing agents like NADH and NADPH in this way because they serve as markers of cellular metabolic activity in the subsequent investigations. Using NADH and NADPH as electron donors enables dye reduction through metabolic reduction, resulting in a noticeable color change (Prabst et al., 2017). Measuring a biochemical marker is the basis of colorimetric tests, which evaluate cells' metabolic capabilities. Colorimetric assays use chemicals that change color depending on the cell's viability; this allows a “spectrophotometer” to detect whether the cells are alive. For cell lines that are “adherent” or “suspended,” “colorimetric assays” are viable options. Both the procedure and the cost are straightforward and affordable (Prabst et al., 2017). Colorimetric assays typically exhibit a consistent pattern in their methodology. We started by cultivating cancer cell lines in a 96-well plate and then introduced reagents to the plate. The next step involves placing the well plate in the incubator for the required duration. Next, a spectrophotometer measures the absorbance at the designated wavelength for each conducted assay. The cell seeding concentration in all assays is chosen as per the requirement of the optimized protocol, which could vary from one experiment to another.

2.2.1. MTT assay

The MTT assay, initially introduced by Mosmann in 1983 and subsequently refined over time by Niks and Otto in 1990, is a highly sensitive colorimetric technique. It enables accurate quantitative measurements of cell viability, proliferation, and activation. The test requires the enzyme cellular mitochondrial dehydrogenase to transform the yellow substance “3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide” (MTT) into a dark blue/purple formazan product that remains insoluble in water. The amount of formazan produced is directly proportional to the number of cells across various cell lines (Mosmann, 1983; Gerlier and Thomasset, 1986; Vega-Avila and Pugsley, 2011). You can take readings using either a spectrophotometer or a microplate reader after breaking up the formazan crystals in a good solvent like DMSO or isopropyl alcohol (Milherio et al., 2016; Sahin, 2023). A readily available kit or a pure “tetrazolium salt” called “thiazolyl blue tetrazolium bromide” are the two ways that MTT can be acquired. That salt can be dissolved and kept for further use. Store the light-sensitive MTT reagent stock solution in the dark at a temperature of -20 °C. Refreezing thawed portions is crucial to prevent the formation of formazan due to unclear MTT conversion (Sylvester, 2011; Prabst et al., 2017). The MTT assay methodology involves several steps, the first of which involves dissolving the salt in 7.4 pH Dulbecco's phosphate-buffered saline (DPBS) to create a 5 mg/ml solution of MTT. Before being transferred into a sterile container that is protected from light, this solution is filtered and sterilized using a 0.2-µm filter (Kamiloglu et al., 2020). Every well of a 96-well plate is seeded with 6,000 cells, and then the cells are placed in a CO2 incubator set at 37°C for 24 hours while the test chemicals are present. The concentration is not fixed as it changes with the requirement of an optimized protocol. The next step is to replace the old media with newer ones. After a 72-hour incubation period, each well is incubated for 1 to 4 hours after adding 20 μL of the 5 mg/mL MTT solution. After the treatment medium was taken out, 100μL of DMSO, a substance-solvent solution, was put into each well to break up the formazan crystals. An ELISA reader was utilized to measure the absorbance at 570 nm. The ability of living cells to absorb the purple formazan that is made when the MTT reagent is broken down was used to check if the cells were still alive. For each cell line, a total of three independent experiments were performed, with each experiment being repeated three times in triplicate (Cai et al., 2008; Gul-e-Saba et al., 2014; Mahar et al., 2020; Mehmood et al., 2022).

2.2.2. MTS assay

In the MTS assay, living cells subject the MTS compound to bio-reduction in the presence of phenazine methosulfate (PMS), forming a soluble formazan product in the culture medium. It is believed that metabolically active cells include NADPH-dependent dehydrogenase enzymes that carry out the conversion process, creating the formazan dye. Cory et al. (1991) measured the absorbance of this dye between 490 and 500 nm. The MTS test, also known as a “one-step” MTT assay, enables direct addition of the reagent to cell cultures, eliminating the need for the periodic steps typically required in the MTT assay. MTS is non-toxic and quite soluble, so it can safely reintroduce cells into the culture, unlike TXT. To be effective, MTS, like XTT, needs PMS (Cory et al., 1991; Kuete et al., 2017). To create the MTS solution, dissolve 2 mg/ml of MTS powder in DPBS until a yellow and transparent solution emerges. Store the MTS solution at -20 degrees Celsius until it's ready for analysis. If immediate use is required, store it at 4°C. Protecting the solution from light is crucial (Kamiloglu et al., 2020). The methodology begins by seeding the cells in the 96-well plates with the desired concentration, specifically six thousand cells per well. Then, for a full day, the test chemicals and the specimens were placed in an incubator set at 37°C in a humidified environment that contained 5% (v/v) CO₂ (Masood et al., 2019, 2023). Once the cell growth reached 80% confluency, we removed the culture medium and replaced it with a new one. Add 20 μL of MTS reagent at the beginning of each incubation phase that lasted between one and four hours at 37 °C. Finally, an ELISA reader was used to detect the absorbance at a wavelength of 490 nm (Chaudhry et al., 2019a, b, 2021a, b).

2.2.3. WST-8 assay

Tominaga synthesized a tetrazolium salt of the second generation, known as “WST-8,” in 1999. Most of the time, the dye can't cross the cell membrane because of its negative net charge. To assess WST-8 sustainability, we need to use an intermediary electron acceptor to facilitate its reduction outside of cells. Finding the absorbance in the culture medium at a wavelength of 450 nm allows one to quantify the level of decreased WST-tetrazolium. Berridge et al. (2005) state that this enables the execution of tests in real-time. Researchers have established a direct correlation between the number of viable cells and the decline in dye concentration. This is an accurate prediction for cells undergoing exponential development. However, complications arise when we exhaust nutrients or test substances that impact metabolic activity. Therefore, it is necessary to establish ideal culture conditions and conduct a comprehensive calibration using the specific cell lines and culture method of interest. This calibration is essential for evaluating the linear range and the relationship between cell concentration and formazan absorbance (Weyermann et al., 2005; Prabst et al., 2017). The WST-8 reagent solution is made up of exactly 5 mM WST-8, 0.2 mM 1-methoxy PMS, and 150 mM NaCl, all of which are dissolved in water. Cell suspensions are carefully placed in 96-well plates, with each well containing 100 µl of test compounds. These plates are then placed in a controlled environment with a temperature of 37°C and a humidity level of 5% CO2 to ensure optimal conditions for the cells. The cells are left to incubate for the necessary amount of time to achieve the desired exposure. Next, a volume of 10 µL of the WST-8 reagent solution is carefully added to each well. Next, set the plate in an incubator at 37°C and leave it undisturbed for 2 hours. After incubation, a multiplate reader quantifies the absorbance at 450 nm (Kamiloglu et al., 2020).

2.2.4. SRB assay

The SRB assay, initially introduced by Skehan and colleagues, evolved for implementation in the National Cancer Institute's extensive program focused on discovering anticancer drugs. The National Cancer Institute initiated this disease-oriented initiative in 1985. SRB is a vibrant pink “aminoxanthene dye” that contains two sulfonic groups. In cells that have been fixed with trichloroacetic acid, SRB binds to basic amino acid residues in proteins when the environment is slightly acidic. This interaction serves as a highly sensitive indicator of cellular protein levels. Researchers commonly employ the SRB assay to evaluate the formation or extinction of colonies (Skehan et al., 1990; Aslanturk, 2018). Researchers specifically design this assay for manual or semi-automatic testing. It offers a highly efficient and sensitive method for testing chemotherapeutic drugs or small molecules in adherent cells. It also has practical applications in assessing the impact of manipulating gene expression (such as reducing or increasing it) and investigating how replacing miRNA affects cell growth (Kasinski et al., 2015). The suggested method has been enhanced to optimize the assessment of drug toxicity on adherent cells in a 96-well format. After the incubation period, we treated a layer of cells with a 10% (wt/vol) solution of trichloroacetic acid and allowed them to stain for 30 minutes. The surplus dye was subsequently eliminated by continuously rinsing the cells with a 1% (vol/vol) solution of acetic acid. The dye that was attached to the protein was mixed with 10 mM Tris base so that its optical density (OD) at 510 nm could be measured with a microplate reader (Vichai and Kirtikara, 2006).

2.2.5. XTT assay

The XTT reduction assay is one of the colorimetric methods for semiquantitatively assessing metabolically active mitochondrial activity. Through the work of mitochondrial succinate dehydrogenase, a yellow XTT salt called “(2,3-bis (2-methoxy-4-nitro-5-sulfophenyl)-5[(phenylamino)carbonyl]-2H-tetrazolium hydroxide)” changes into an orange water-soluble formazan through mitochondrial succinate dehydrogenase (Roehm et al., 1991). The number of metabolically active microbial cells can be determined by measuring the variations in color caused by the reaction between cells and XTT salt. This measurement can be done using spectrophotometry. “Mitochondrial dehydrogenases” are enzymes that are found in metabolically active cells. They lower the “tetrazolium salt” (XTT) on the outside, which makes colorimetric formazans (Antachopoulos et al., 2006; Moss et al., 2008). This phenomenon arises due to the transfer of electrons across the plasma membrane by NADH, which is generated during the tricarboxylic acid cycle. This electron transfer reduces menadione (Berridge et al., 2005). Consequently, the reduction of menadione enables the formation of colorimetric formazan, which in turn allows for the quantification of metabolic activity in a microbial culture utilizing spectrophotometry (Moss et al., 2008; Moffa et al., 2016). The biofilm-coated wells were washed twice with sterile PBS to eliminate any cells that did not attach. After that, 160µL of sterile PBS and 40µL of XTT salt solution with 1% phenazine methosulphate (Sigma-Aldrich, USA) were added to each well until the total volume reached 200µL. The plate was incubated at 37°C for three hours. After the incubation period, 100 μL of the suspension was moved to a second, clean 96-well plate, and the microwave reader was used to measure the absorbance at 450 nm and 620 nm. Arzmi et al. (2023) measured cell viability using a reference wavelength of 620 nm. The absorbance at 450 nm and subtracted it from the absorbance at 620 nm.

2.2.6. WST-1 assay

5-(2,4-disulfophenyl) The chemical name of WST-1 is -2-(4-nitrophenyl)-2-(4-iodophenyl). The monosodium salt -2H-tetrazolium assay is a popular colorimetric method for determining cell viability. In theory, WST-1 works the same way as MTT; it reacts with mitochondrial succinate tetrazolium reductase to make a formazan dye that is water-soluble (Nemudzivhadi and Masoko, 2014). The net negative charge of WST-1, which is prevented from entering cells by its two sulfonate groups, is different. Tetrazolium salts with negative charges, such as WST-1, undergo reduction outside of cells due to their inability to enter them. An intermediate electron carrier facilitates the transfer of electrons over the plasma membrane, allowing for the extracellular reduction to take place (Berridge et al., 2005; Sari et al., 2021). Before adding the test chemical at the appropriate dose, culture the cells in 96-well plates until they reach 80–90% confluency. The experiment calls for incubating the cells at 37°C. To get a final concentration of 0.33 mg/L, add WST solution to each well. Depending on the cell type and the experiment, incubate at 37°C in the dark for 30 minutes to 2 hours. Use a microplate reader to measure absorbance between 420 and 480 nm (Sumantran, 2011).

2.2.7. CVS assay

Crystal violet staining (CVS) involves using a triphenylmethane dye known as crystal violet, also referred to as “gentian violet” or “hexamethyl pararosaniline chloride” (Castro-Garza et al., 2007). Initially used to count cells in monolayer cultures by measuring dye absorption (Gillies et al., 1986), the CVS assay has evolved and is now utilized for various purposes. These include assessing the cytotoxic effects of chemicals, drugs, or toxins on cells to determine viability (Thomas et al., 2004) or evaluating cell growth across different experimental conditions (Zivadinovic et al., 2005). The CVS assay is a straightforward method that provides quantitative data on the relative density of cells adhering to multi-well cluster dishes. It is important to be aware that this dye could color DNA, and the specific hue of the dye is determined by the pH level of the solution. The amount of dye absorbed by the monolayer and the strength of the resulting color are directly related to the number of cells when the dye is dissolved (Naseer et al., 2009; Vega-Avila and Pugsley, 2011). The brief methodology is as follows: After the biofilm incubation was completed, the 96-well plate was rinsed twice by carefully removing 200 μL of suspension from each well. Afterwards, careful pipetting of 200 μL of sterile PBS into each well was done twice to eliminate any cells that failed to adhere. Afterward, fix each well with 200 μL of methanol and incubate at 25°C for 15 minutes. The dish was allowed to air dry for half an hour after the liquid was drained. Following that, 200 μL of a CVS solution containing 0.1% (w/v) was added to every well and allowed to incubate at 25°C for a duration of 20 minutes. Next, we used distilled water to wash the plate twice. To eliminate the biofilm stains, 200 μL of acetic acid, which is 33% (v/v) concentrated, was used. For 5 minutes, the plate was allowed to incubate at room temperature. A microplate reader was used to measure the absorbance at OD (620 nm) after 100 μL of this solution was moved to a new, clean 96-well plate. The procedure was triple tested in terms of both biological and technological aspects (Arzmi et al., 2023).

2.3. Fluorometric assays

“Fluorometric assays” are simple to do with tools like a “fluorescence microscope,” “fluorometer,” “fluorescence microplate reader,” or “flow cytometer.” In many ways, they are better than “dry exclusion” and “colorimetric assays.” These methods work better than colorimetric ones for both “adherent” and “suspended cell lines” (O'Brien et al., 2000; Page et al., 1993; Aslanturk, 2018). These assays rely on the enzymatic reaction between a substrate and enzyme to produce a luminescent molecule. The enzyme and substrate concentrations have a direct impact on this molecule's synthesis rate. To get a quantitative idea of the reaction rate, you can speed up the production of the luminescent compound and use a fluorometer to measure the amount of fluorescence released per unit of time (Guilbault, 1972). Page et al. (1993) used the Alamar Blue assay, a fluorometric technique, to measure the metabolic activity of cells. The method relies on mitochondrial enzymes with diaphorase activity, like “NADPH dehydrogenase” (O'Brien et al., 2000), to change resazurin (the oxidized form; 7-hydroxy-3H-phenoxazin-3-1-10-oxide) to resorufin (the reduced form). Visually, the blue and weakly emitting resazurin undergoes a slow conversion by cells into the red, strongly emitting resorufin (Zachari et al., 2013). The alamar blue method is a very sensitive, uncomplicated, and secure technique for monitoring cell survival and growth (Larson et al., 1997). “Resazurin” is a “phenoxazin-3-one” dye and a redox indicator that can pass through cells. It is often used in scientific methods to check the number of living cells, such as “tetrazolium compounds” methods (Ahmed et al., 1994). It functions as a mediator in the electron transport chain, taking on the role of an electron acceptor in place of molecular oxygen, eventually facilitating the reduction of oxygen and cytochrome oxidase (Page et al., 1993). This compound is safe to use and can easily enter cells. The compound exhibits a blue color and lacks fluorescence. Once it enters the cells, resazurin is reduced to “resorufin.” The compound “resorufin” exhibits a vibrant red color and possesses a remarkable level of fluorescence. Cells that are alive can convert resazurin into resofurin, which leads to an increase in fluorescence and color in the medium they are growing in. The number of viable cells influences the production of resofurin (Aslanturk, 2018). The methodology includes the following steps: Once the 96-well plates have been prepared for the experiment, proceed to measure the volume of the culture medium in each well. Measure out 0.1× volume of alamar blue and add it to the well. Allow the sample to rest for 1–4 hours at a temperature of 37°C in a controlled environment. Dispense 100 µL of the cell culture supernatant into a flat-bottomed, 96-well plate. Choose either Step 5 or Step 6 to continue. If the reading is not performed immediately, it is important to pause and ensure the accuracy of the results. This can be achieved by adding 50µL of 3% SDS per 100µL of the original culture volume, which helps to stabilize the action. Store the plate at the appropriate temperature for a maximum of 24 hours before proceeding. To avoid evaporation, it is critical to shield the contents from light and cover them adequately. Measure the plate using an excitation wavelength of 560 nm and an emission wavelength of 590 nm. When using a fixed-wavelength plate reader, one can utilize an excitation range of 540–570 nm and an emission range of 580–610 nm (Kumar et al., 2018).

2.3.1. CFDA-AM assay

“CFDA-AM” is a fluorogenic dye commonly employed in cytotoxicity studies to assess plasma membrane integrity. This dye, CFDA-AM, undergoes a transformation when exposed to esterases in living cells. It changes from a nonpolar, nonfluorescent substance to a polar, fluorescent dye called carboxyfluorescein (CF). The esterases that are present in the cells facilitate this process. The conversion to CF by the cells demonstrates the importance of an unaltered plasma membrane in maintaining the cytoplasmic milieu necessary for esterase activity (Ganassin et al., 2000; Bopp and Lettieri, 2008). The methodology involves treating each cell with a four-M solution of CFDA-AM after removing the adherent cells' growth media from the wells. For suspension cell cultures, we dilute a 4 mM stock solution in serum- and amino acid-free culture media at a ratio of 1:500 to create an 8 mM CFDA-AM working solution. Next, we transfer the cells to the wells using serum- and amino acid-free culture media. To each well, an equal volume of the 8 μM CFDA-AM working solution is added. To start the 96-well plate cultures, 100 μL of growth medium free of serum and amino acids is put into each well. This is followed by 100 μL of the 8 μM CFDA-AM working solution. The cells are incubated with CFDA-AM at 18–22 °C for 30 minutes while remaining in darkness. To find the fluorescence, a fluorescent plate reader is used. The excitation wavelength is 493 nm, and the emission wavelength is 541 nm after incubation (Ganassin & Bols, 2000; Schirmer et al., 1997; Schreer et al., 2005; Kamiloglu et al., 2020).

2.3.2. Dye exclusion assay

The dye exclusion assay is a common method for measuring the number of viable cells in a cell suspension. It is based on the principle that live cells have intact cell membranes that prevent certain dyes, like trypan blue, eosin, or propidium, from entering, while dead cells do not (Strober, 2001). These assays rely on the observation that live cells with intact plasma membranes exclude certain dyes, while dead cells do not.

2.3.3. Trypan blue assay

The trypan blue dye exclusion assay is a commonly employed method in scientific research to assess cell viability. Various studies (Avelar-Freitas et al., 2014; Tran et al., 2011) have widely used it. The process includes using trypan blue to stain deceased cells and then examining them under a microscope using a hemocytometer (Avelar-Freitas et al., 2014). In 1975, Kamiloglu et al. developed this method to quantify living cells and provide insights into cell death. Trypan blue is a non-permeable dye used to stain cell membranes. It is derived from toluidine and belongs to the azo dye family. This stain is commonly employed in the field of bioscience to selectively stain cells that have undergone necrosis, indicating cell death. Thus, this assay relies on the idea that viable cells have intact cell membranes that prevent trypan blue penetration. As a result, trypan blue is unable to enter viable cells (Jain et al., 2018). On the other hand, the trypan blue dye can enter the cytoplasm of necrotic (dead) cells because their cell membrane has lost its integrity. When observed under a light microscope, it is interesting to note that only necrotic (dead) cells exhibit an absorption of this blue color, as mentioned in studies conducted by Chan et al. (2020) and Ude et al. (2022). Centrifugation of the sample is necessary to determine the viability of a cell suspension. Instruct the centrifuge to spin at 100 Å g for 5 minutes. After centrifugation, remove the liquid that floats on top of the particles. To resuspend the cell pellet, use 1 milliliter of PBS or serum-free complete medium. Make a diluted cell combination by combining cell suspension with equal parts of 0.4% trypan blue. Set aside the combination at room temperature for about three minutes. Use a “hemacytometer” to transfer a tiny amount of the trypan blue/cell sample. Put the “hemacytometer” on a binocular microscope's stage and focus it so you can see the cells. Use a “hemacytometer” to separate viable (unstained) and nonviable (stamped) cells. Multiplying the overall number of viable cells by 2, the trypan blue dilution factor, yields the total number of cells per milliliter of aliquot. Add the numbers of live and dead cells in an aliquot, then multiply that amount by 2 to get the cell count per milliliter (Strober, 2001).

2.3.4. Propidium Iodide (PI) stain

“Propidium iodide” is an additional DNA dye that can be used for cell quantization. For this red DNA stain to enter the cell, the cell membrane needs to be permeabilized, as the dye cannot pass through the intact cell membrane. Propidium iodide is classified as a compound in the phenanthridinium group. It can interact with both “double-stranded DNA” and “RNA” without showing any preference for nucleosides (Quent et al., 2010; Blaheta et al., 1998). Propidium iodide exhibits a significant increase in fluorescence when it is bound to DNA (Godinho et al., 2018). The protocol's sensitivity for measuring the propidium iodide signal may range from 150 to 500 cells. Despite the protocol's high sensitivity, the primary purpose of this dye is to assess cell viability (Ligasova and Koberna, 2021). To conduct the PI staining, the cell seeding followed by staining occurred. The density of cells sown in a 96-well microtitre plate was 1 × 10⁴ cells per well. The test substances were applied to the cells. Each well was treated with 1 µl of propidium iodide (PI) reagent, which had a concentration of 0.5 mg/ml, and then allowed to incubate at room temperature for 10 minutes. To measure the dual fluorescence, a multi-detection microplate reader was used. The study by Hussain et al. (2019) used the 525 nm excitation and 595 nm PI emission wavelengths. Table 1 provides a brief overview of all assays and methodology.

Table 1
The list of cytotoxicity assay types description and methodology.

Serial No	Assay Name	Description	Brief Methodology	References
1	MTT	MTT is converted to formazan crystals by mitochondrial enzymes in living cells, which are solubilized and quantified using spectrophotometry.	Incubate cells with MTT solution for 2-4 hours, then solubilize formazan crystals with DMSO. Measure absorbance at 570 nm using a microplate reader.	Mosmann (1983)
2	MTS	A soluble formazan product is formed in the culture medium when the MTS compound is subjected to bio-reduction by living cells in the presence of phenazine methosulfate (PMS).	To produce the MTS solution, dissolve 2 mg/ml of MTS powder in DPBS until a yellow and transparent solution is obtained. Keep the MTS solution at -20 degrees Celsius until fully prepared. If immediate use is necessary, it can also be stored at 4°C. Protect the solution from light. Add 20 μL of MTS reagent at the beginning of each incubation phase lasting between one and four hours at 37°C. Measure absorbance at 490 nm using an ELISA reader.	Cory et al. (1991); Kuete et al. (2017)
3	WST-8	WST-8 is reduced outside of cells, and the absorbance in the culture medium is measured at a wavelength of 450 nm to quantify the level of decreased WST-tetrazolium.	Prepare WST-8 reagent solution containing 5 mM WST-8, 0.2 mM 1-methoxy PMS, and 150 mM NaCl dissolved in water. Incubate cell suspensions in 96-well plates at 37°C. Add 10 µL of WST-8 reagent solution to each well and incubate for 2 hours. Measure absorbance at 450 nm using a multiplate reader.	Berridge et al. (2005); Sari et al. (2021)
4	SRB	The SRB assay utilizes a vibrant pink dye that binds to basic amino acid residues in proteins in fixed cells.	Fix cells with 10% trichloroacetic acid and stain with 0.1% SRB solution. Wash with acetic acid, and measure optical density at 510 nm using a microplate reader.	Skehan et al. (1990); Aslanturk (2018)
5	XTT	XTT salt is reduced by metabolically active cells, forming an orange formazan dye, which is quantified using spectrophotometry.	Wash biofilm-coated wells with sterile PBS and add XTT salt solution with 1% phenazine methosulphate. Incubate at 37°C for three hours, then measure absorbance at 450 nm and 620 nm. Subtract absorbance at 450 nm from absorbance at 620 nm for cell viability assessment.	Roehm et al. (1991); Moss et al. (2008)
6	WST-1	Similar to MTT, WST-1 produces a water-soluble formazan dye, which is quantified using a microplate reader.	Culture cells in 96-well plates and add WST-1 solution. Incubate at 37°C and measure absorbance at 420-480 nm.	Nemudzivhadi and Masoko (2014); Sumantran (2011);
7	CVS	CVS staining involves a triphenylmethane dye that binds to basic amino acid residues in proteins in fixed cells.	Incubate cells with crystal violet solution, wash with acetic acid, and measure absorbance at OD 620 nm.	Castro-Garza et al. (2007); Naseer et al. (2009)
8	Resazurin	Resazurin is converted to resorufin by mitochondrial enzymes in living cells, leading to an increase in fluorescence.	Add resazurin to cell culture and incubate. Measure fluorescence using excitation wavelength of 560 nm and emission wavelength of 590 nm.	Page et al. (1993); Larson et al. (1997)
9	CFDA-AM	CFDA-AM is transformed into a fluorescent dye called carboxyfluorescein (CF) by esterases in living cells.	Treat cells with CFDA-AM solution and incubate. Measure fluorescence using excitation wavelength of 493 nm and emission wavelength of 541 nm.	Ganassin et al. (2000); Schirmer et al. (1997)
10	Dye exclusion	Live cells exclude certain dyes due to intact cell membranes, while dead cells do not.	Centrifuge cell suspension, resuspend pellet in PBS, and mix with trypan blue. Count live and dead cells under a microscope using a hemocytometer.	Strober (2001); Jain et al. (2018)
11	Trypan blue	Dead cells are stained with trypan blue, which cannot penetrate intact cell membranes.	Centrifuge cell suspension, resuspend pellet in PBS, mix with trypan blue, and incubate. Count live and dead cells under a microscope using a hemocytometer.	Kamiloglu et al. (2020); Chan et al. (2020)
12	Propidium Iodide stain	Propidium iodide is used to assess cell viability by binding to DNA in cells with permeabilized membranes.	Treat cells with propidium iodide solution and incubate. Measure fluorescence using a multi-detection microplate reader with excitation wavelength of 525 nm and emission wavelength of 595 nm.	Quent et al. (2010); Blaheta et al. (1998)

3. Conclusion and Future Recommendations

Assays for determining cell cytotoxicity are considered important in the development of new drugs. While choosing an assay for in vitro cytotoxicity, there are some crucial factors that need to be considered, including speed, safety, reliability, efficacy, and cost-efficiency. It is important to select an assay method that does not interfere with the test compound and can effectively assess the nature of the interaction. The choice of cell line can have a significant impact on the assay's results. Therefore, it is crucial to thoroughly test and assess different methods before settling on a final selection. If order to improve the reliability of the results obtained, it is advisable to use multiple assays, if possible. Given these factors, it is advisable to thoroughly assess the various assay methods and choose the one that most effectively fulfils the desired criteria. It is important to consider the test compound's mechanism of action and select an assay method that is compatible with it.

Acknowledgements

The authors would like to acknowledge the research is funded by Fundamental Research Grant Scheme, Ministry of Higher Education Malaysia (FRGS/1/2024/STG01/UMT/02/2)

References

• GUL-E-SABA, ABDAH, A., and ABDULLAH, M. Cytotoxic effects of drug-loaded hyaluronan-glutaraldehyde cross-linked nanoparticles and the release kinetics modeling. Journal of Advanced Chemical Engineering, vol. 1, no. 104, pp. 2.
• ADAN, A., KIRAZ, Y. and BARAN, Y., 2016. Cell proliferation and cytotoxicity assays. Current Pharmaceutical Biotechnology, vol. 17, no. 14, pp. 1213-1221. http://doi.org/10.2174/1389201017666160808160513 PMid:27604355.
  » http://doi.org/10.2174/1389201017666160808160513
• AHMED, S.A., GOGAL JUNIOR, R.M.J. and WALSH, J.E., 1994. A new rapid and simple non-radioactive assay to monitor and determine the proliferation of lymphocytes: an alternative to [3H]thymidine incorporation assay. Journal of Immunological Methods, vol. 170, no. 2, pp. 211-224. http://doi.org/10.1016/0022-1759(94)90396-4 PMid:8157999.
  » http://doi.org/10.1016/0022-1759(94)90396-4
• ANTACHOPOULOS, C., MELETIADIS, J., ROILIDES, E., SEIN, T. and WALSH, T.J., 2006. Rapid susceptibility testing of medically important zygomycetes by XTT assay. Journal of Clinical Microbiology, vol. 44, no. 2, pp. 553-560. http://doi.org/10.1128/JCM.44.2.553-560.2006 PMid:16455912.
  » http://doi.org/10.1128/JCM.44.2.553-560.2006
• ARZMI, M.H., KAMIL, W.N.W.A. and ZAINAL, M., 2023. Monospecies and polymicrobial biofilms in static and flow environment. Methods in Microbiology, vol. 53, pp. 51-66. http://doi.org/10.1016/bs.mim.2023.05.002
  » http://doi.org/10.1016/bs.mim.2023.05.002
• ASLANTURK, Ö.S. 2018. In vitro cytotoxicity and cell viability assays: principles, advantages, and disadvantages. In M. L. LARRAMENDY and S. SOLONESKI, editors. Genotoxicity: a predictable risk to our actual world Londres: IntechOpen, vol. 2, pp. 64-80.. http://doi.org/10.5772/intechopen.71923
  » http://doi.org/10.5772/intechopen.71923
• AVELAR-FREITAS, B.A., ALMEIDA, V.G., PINTO, M.C., MOURAO, F.A., MASSENSINI, A.R., MARTINS-FILHO, O.A., ROCHA-VIEIRA, E. and BRITO-MELO, G.E., 2014. Trypan blue exclusion assay by flow cytometry. Brazilian Journal of Medical and Biological Research, vol. 47, no. 4, pp. 307-315. http://doi.org/10.1590/1414-431X20143437 PMid:24652322.
  » http://doi.org/10.1590/1414-431X20143437
• BERRIDGE, M.V., HERST, P.M. and TAN, A.S., 2005. Tetrazolium dyes as tools in cell biology: new insights into their cellular reduction. Biotechnology Annual Review (Amsterdam), vol. 11, pp. 127-152. http://doi.org/10.1016/S1387-2656(05)11004-7 PMid:16216776.
  » http://doi.org/10.1016/S1387-2656(05)11004-7
• BLAHETA, R.A., KRONENBERGER, B., WOITASCHEK, D., WEBER, S., SCHOLZ, M., SCHULDES, M., ENCKE, A. and MARKUS, B.H., 1998. Development of an ultrasensitive in vitro assay to monitor growth of primary cell cultures with reduced mitotic activity. Journal of Immunological Methods, vol. 211, no. 1-2, pp. 159-169. http://doi.org/10.1016/S0022-1759(97)00202-0 PMid:9617840.
  » http://doi.org/10.1016/S0022-1759(97)00202-0
• BOPP, S.K. and LETTIERI, T., 2008. Comparison of four different colorimetric and fluorometric cytotoxicity assays in a zebrafish liver cell line. BMC Pharmacology, vol. 8, no. 1, pp. 8. http://doi.org/10.1186/1471-2210-8-8 PMid:18513395.
  » http://doi.org/10.1186/1471-2210-8-8
• CAI, S., XIE, Y., BAGBY, T.R., COHEN, M.S. and FORREST, M.L., 2008. Intralymphatic chemotherapy using a hyaluronan-cisplatin conjugate. The Journal of Surgical Research, vol. 147, no. 2, pp. 247-252. http://doi.org/10.1016/j.jss.2008.02.048 PMid:18498877.
  » http://doi.org/10.1016/j.jss.2008.02.048
• CASTRO-GARZA, J., BARRIOS-GARCIA, H.B., CRUZ-VEGA, D.E., SAID-FERNANDEZ, S., CARRANZA-ROSALES, P., MOLINA-TORRES, C.A. and VERA-CABRERA, L., 2007. Use of a colorimetric assay to measure differences in cytotoxicity of Mycobacterium tuberculosis strains. Journal of Medical Microbiology, vol. 56, no. 6, pp. 733-737. http://doi.org/10.1099/jmm.0.46915-0 PMid:17510256.
  » http://doi.org/10.1099/jmm.0.46915-0
• CHAN, L.L., RICE, W.L. and QIU, J., 2020. Observation and quantification of the morphological effect of trypan blue rupturing dead or dying cells. PLoS One, vol. 15, no. 1, pp. e0227950. http://doi.org/10.1371/journal.pone.0227950 PMid:31978129.
  » http://doi.org/10.1371/journal.pone.0227950
• CHANNAR, P.A., AZIZ, M., EJAZ, S.A., CHAUDHRY, G.E., SAEED, A., UJAN, R., HASAN, A. and SAEED, A., 2023. Structural and functional insight into thiazolidinone derivatives as novel candidates for anticancer drug design: in vitro biological and in-silico strategies. Journal of Biomolecular Structure & Dynamics, vol. 41, no. 3, pp. 942-953. http://doi.org/10.1080/07391102.2021.2018045 PMid:34927557.
  » http://doi.org/10.1080/07391102.2021.2018045
• CHAUDHRY, G., KASSIM, M.N. and ZAFAR, M., 2020. Induction of apoptosis by Stichopus chloronotus and Holothuria nobilis fractions in the human cervical cancer cell line, HeLa. International Journal of Research in Pharmaceutical Sciences, vol. 11, no. 1, pp. 1238-1247. http://doi.org/10.26452/ijrps.v11i1.1964
  » http://doi.org/10.26452/ijrps.v11i1.1964
• CHAUDHRY, G.E., ISLAMIAH, M., ZAFAR, M.N., BAKAR, K., AZIZ, N., SAIDIN, J., SUNG, Y.Y. and MUHAMMAD, T.S.T., 2021a. Induction of apoptosis by Acanthaster planci sp., and Diadema setosum sp., fractions in human cervical cancer cell line, HeLa. Asian Pacific Journal of Cancer Prevention, vol. 22, no. 5, pp. 1365-1373. http://doi.org/10.31557/APJCP.2021.22.5.1365 PMid:34048163.
  » http://doi.org/10.31557/APJCP.2021.22.5.1365
• CHAUDHRY, G.E., JAN, R., AKIM, A., ZAFAR, M.N., SUNG, Y.Y. and MUHAMMAD, T.S.T., 2021c. Breast cancer: a global concern, diagnostic and therapeutic perspectives, mechanistic targets in drug development. Advanced Pharmaceutical Bulletin, vol. 11, no. 4, pp. 580-594. http://doi.org/10.34172/apb.2021.068 PMid:34888205.
  » http://doi.org/10.34172/apb.2021.068
• CHAUDHRY, G.E., JAN, R., MOHAMAD, H. and MUHAMMAD, T.S.T., 2019a. Vitex rotundifolia fractions induce apoptosis in human breast cancer cell line, MCF-7, via extrinsic and intrinsic pathways. Research in Pharmaceutical Sciences, vol. 14, no. 3, pp. 273-285. http://doi.org/10.4103/1735-5362.258496 PMid:31160905.
  » http://doi.org/10.4103/1735-5362.258496
• CHAUDHRY, G.E., JAN, R., ZAFAR, M.N., MOHAMMAD, H. and MUHAMMAD, T.S.T., 2019b. Vitex Rotundifolia fractions induced apoptosis in human breast cancer T-47D cell line via activation of extrinsic and intrinsic pathway. Asian Pacific Journal of Cancer Prevention, vol. 20, no. 12, pp. 3555-3562. http://doi.org/10.31557/APJCP.2019.20.12.3555 PMid:31870094.
  » http://doi.org/10.31557/APJCP.2019.20.12.3555
• CHAUDHRY, G.E., MD AKIM, A., SUNG, Y.Y. and SIFZIZUL, T.M.T., 2022a. Cancer and apoptosis: the apoptotic activity of plant and marine natural products and their potential as targeted cancer therapeutics. Frontiers in Pharmacology, vol. 13, pp. 842376. http://doi.org/10.3389/fphar.2022.842376 PMid:36034846.
  » http://doi.org/10.3389/fphar.2022.842376
• CHAUDHRY, G.E., ZEENIA, AKIM, A.M. and MUUHAMMAD, T., 2024c. Comprehensive review on mechanistic insights, optimal dosages, and safety prospective of natural products in anticancer therapeutics. Food and Drug Safety, vol. 1, no. 1, pp. 10-27. http://doi.org/10.55121/fds.v1i1.137
  » http://doi.org/10.55121/fds.v1i1.137
• CHAUDHRY, G.E.S., AKIM, A.M., SUNG, Y.Y. and MUHAMMAD, T.S.T., 2022b. Cancer and apoptosis. In: H. BARCENILLA and D. DIAZ, eds. Apoptosis and cancer: methods and protocols New York, NY: Springer US, pp. 191-210.. http://doi.org/10.1007/978-1-0716-2553-8_16
  » http://doi.org/10.1007/978-1-0716-2553-8_16
• CHAUDHRY, G.E.S., SOHIMI, N.K.A., MOHAMAD, H., ZAFAR, M.N., AHMED, A., SUNG, Y.Y. and MUHAMMAD, T.S.T., 2021b. Xylocarpus moluccensis induces cytotoxicity in human hepatocellular carcinoma HepG2 cell line via activation of the extrinsic pathway. Asian Pacific Journal of Cancer Prevention, vol. 22, no. 1, pp. 17-24. http://doi.org/10.31557/APJCP.2021.22.S1.17 PMid:33576208.
  » http://doi.org/10.31557/APJCP.2021.22.S1.17
• CHAUDHRY, G.E.S., ZEENIA, X., AKIM, A.M., SUNG, Y.Y. and MUHAMMAD, T.S.T., 2024b. Green and Blue solutions: natural compounds from plants and marine organisms therapeutic role in induction of cancer cell death. In: N. REZAEI, ed. Interdisciplinary cancer research Cham: Springer, pp. 1-31.
• CHAUDHRY, G.E.S., ZEENIA., SHARIFI-RAD, J. and CALINA, D., 2024a. Hispidulin: a promising anticancer agent and mechanistic breakthrough for targeted cancer therapy. Naunyn-Schmiedeberg’s Archives of Pharmacology, vol. 397, no. 4, pp. 1919-1934. http://doi.org/10.1007/s00210-023-02645-9 PMid:37594522.
  » http://doi.org/10.1007/s00210-023-02645-9
• CORY, A.H., OWEN, T.C., BARLTROP, J.A. and CORY, J.G., 1991. Use of an aqueous soluble tetrazolium/formazan assay for cell growth assays in culture. Cancer Communications, vol. 3, no. 7, pp. 207-212. http://doi.org/10.3727/095535491820873191 PMid:1867954.
  » http://doi.org/10.3727/095535491820873191
• EISENBRAND, G., POOL-ZOBEL, B., BAKER, V., BALLS, M., BLAAUBOER, B.J., BOOBIS, A., CARERE, A., KEVEKORDES, S., LHUGUENOT, J.C., PIETERS, R. and KLEINER, J., 2002. Methods of in vitro toxicology. Food and Chemical Toxicology, vol. 40, no. 2-3, pp. 193-236. http://doi.org/10.1016/S0278-6915(01)00118-1 PMid:11893398.
  » http://doi.org/10.1016/S0278-6915(01)00118-1
• FOTAKIS, G. and TIMBRELL, J.A., 2006. In vitro cytotoxicity assays: comparison of LDH, neutral red, MTT and protein assay in hepatoma cell lines following exposure to cadmium chloride. Toxicology Letters, vol. 160, no. 2, pp. 171-177. http://doi.org/10.1016/j.toxlet.2005.07.001 PMid:16111842.
  » http://doi.org/10.1016/j.toxlet.2005.07.001
• GANASSIN, R.C. and BOLS, N.C., 2000. Growth of rainbow trout hemopoietic cells in methylcellulose and methods of monitoring their proliferative response in this matrix. Methods in Cell Science, vol. 22, no. 2-3, pp. 147-152. http://doi.org/10.1023/A:1009835814441 PMid:11264947.
  » http://doi.org/10.1023/A:1009835814441
• GANASSIN, R.C., SCHIRMER, K. and BOLS, N.C., 2000. Cell and tissue culture. In: G.K. OSTRANDER, ed. The laboratory fish San Diego: Academic Press, pp. 631-651.. http://doi.org/10.1016/B978-012529650-2/50047-0
  » http://doi.org/10.1016/B978-012529650-2/50047-0
• GARCIA-HERNANDO, M., BENITO-LOPEZ, F. and BASABE-DESMONTS, L., 2020. Advances in microtechnology for improved cytotoxicity assessment. Frontiers in Materials, vol. 7, pp. 582030. http://doi.org/10.3389/fmats.2020.582030
  » http://doi.org/10.3389/fmats.2020.582030
• GERLIER, D. and THOMASSET, N., 1986. Use of MTT colorimetric assay to measure cell activation. Journal of Immunological Methods, vol. 94, no. 1-2, pp. 57-63. http://doi.org/10.1016/0022-1759(86)90215-2 PMid:3782817.
  » http://doi.org/10.1016/0022-1759(86)90215-2
• GILLIES, R.J., DIDIER, N. and DENTON, M., 1986. Determination of cell number in monolayer cultures. Analytical Biochemistry, vol. 159, no. 1, pp. 109-113. http://doi.org/10.1016/0003-2697(86)90314-3 PMid:3812988.
  » http://doi.org/10.1016/0003-2697(86)90314-3
• GODINHO, C.P., PRATA, C.S., PINTO, S.N., CARDOSO, C., BANDARRA, N.M., FERNANDES, F. and SA-CORREIA, I., 2018. Pdr18 is involved in yeast response to acetic acid stress counteracting the decrease of plasma membrane ergosterol content and order. Scientific Reports, vol. 8, no. 1, pp. 7860. http://doi.org/10.1038/s41598-018-26128-7 PMid:29777118.
  » http://doi.org/10.1038/s41598-018-26128-7
• GUILBAULT, G.G., 1972. Newer fluorometric methods for the analysis of biologically important compounds. Journal of Research of the National Bureau of Standards. Section A. Physics and Chemistry, vol. 76, no. 6, pp. 607-612. http://doi.org/10.6028/jres.076A.053 PMid:34565886.
  » http://doi.org/10.6028/jres.076A.053
• GUL, I., YUNUS, U., AJMAL, M., BHATTI, M.H. and CHAUDHRY, G.E., 2021. Development of biodegradable thin films for efficient, specific and controlled delivery of capecitabine. Biomedical Materials (Bristol, England), vol. 16, no. 5, pp. 055019. http://doi.org/10.1088/1748-605X/ac1c61 PMid:34375958.
  » http://doi.org/10.1088/1748-605X/ac1c61
• HE, Y., ZHU, Q., CHEN, M., HUANG, Q., WANG, W., LI, Q., HUANG, Y. and DI, W., 2016. The changing 50% inhibitory concentration (IC₅₀) of cisplatin: A pilot study on the artifacts of the MTT assay and the precise measurement of density-dependent chemoresistance in ovarian cancer. Oncotarget, vol. 7, no. 43, pp. 70803-70821. http://doi.org/10.18632/oncotarget.12223 PMid:27683123.
  » http://doi.org/10.18632/oncotarget.12223
• HUSSAIN, H., AHMAD, S., ABD. RAZAK, M.F., WAN MOHAMUD, W.N., BAKAR, J. and GHAZALI, H.M., 2019. Determination of cell viability using acridine orange/propidium iodide dual-spectrofluorometry assay. Cogent Food & Agriculture, vol. 5, no. 1, pp. 1582398. http://doi.org/10.1080/23311932.2019.1582398
  » http://doi.org/10.1080/23311932.2019.1582398
• ISHIYAMA, M., TOMINAGA, H., SHIGA, M., SASAMOTO, K., OHKURA, Y. and UENO, K., 1996. A combined assay of cell viability and in vitro cytotoxicity with a highly water-soluble tetrazolium salt, neutral red and crystal violet. Biological & Pharmaceutical Bulletin, vol. 19, no. 11, pp. 1518-1520. http://doi.org/10.1248/bpb.19.1518 PMid:8951178.
  » http://doi.org/10.1248/bpb.19.1518
• JAIN, A.K., SINGH, D., DUBEY, K., MAURYA, R., MITTAL, S. and PANDAY, A.K., 2018. Models and methods for in vitro toxicity. In A. DHAWAN ans S. KWON, eds. In vitro toxicology London: Academic Press, pp. 45-65. http://doi.org/10.1016/B978-0-12-804667-8.00003-1
  » http://doi.org/10.1016/B978-0-12-804667-8.00003-1
• JAN, R. and CHAUDHRY, G.E., 2019c. Understanding apoptosis and apoptotic pathways targeted cancer therapeutics. Advanced Pharmaceutical Bulletin, vol. 9, no. 2, pp. 205-218. http://doi.org/10.15171/apb.2019.024 PMid:31380246.
  » http://doi.org/10.15171/apb.2019.024
• KAMILOGLU, S., SARI, G., OZDAL, T. and ÇAPANOGLU GUVEN, E., 2020. Guidelines for cell viability assays. Food Frontiers, vol. 1, no. 3, pp. 332-349. http://doi.org/10.1002/fft2.44
  » http://doi.org/10.1002/fft2.44
• KARATOP, E.U., CIMENCI, C.E. and AKSU, A.M., 2022. Colorimetric cytotoxicity assays. Londres: IntechOpen. http://doi.org/10.5772/intechopen.105772
  » http://doi.org/10.5772/intechopen.105772
• KASINSKI, A.L., KELNAR, K., STAHLHUT, C., ORELLANA, E., ZHAO, J., SHIMER, E., DYSART, S., CHEN, X., BADER, A.G. and SLACK, F.J., 2015. A combinatorial microRNA therapeutics approach to suppressing non-small cell lung cancer. Oncogene, vol. 34, no. 27, pp. 3547-3555. http://doi.org/10.1038/onc.2014.282 PMid:25174400.
  » http://doi.org/10.1038/onc.2014.282
• KUETE, V., KARAOSMANOGLU, O. and SIVAS, H. 2017. Anticancer activities of African medicinal spices and vegetables. In: A. Dhawan, S. Kwon, eds. Medicinal spices and vegetables from Africa London: Academic Press, pp. 271-297.. http://doi.org/10.1016/B978-0-12-809286-6.00010-8
  » http://doi.org/10.1016/B978-0-12-809286-6.00010-8
• KUMAR, P., NAGARAJAN, A. and UCHIL, P.D., 2018. Analysis of cell viability by the alamarBlue assay. Cold Spring Harbor Protocols, vol. 2018, no. 6, pp. pdb.prot095489. http://doi.org/10.1101/pdb.prot095489 PMid:29858336.
  » http://doi.org/10.1101/pdb.prot095489
• KUNZMANN, A., ANDERSSON, B., THURNHERR, T., KRUG, H., SCHEYNIUS, A. and FADEEL, B., 2011. Toxicology of engineered nanomaterials: focus on biocompatibility, biodistribution and biodegradation. Biochimica et Biophysica Acta, vol. 1810, no. 3, pp. 361-373. http://doi.org/10.1016/j.bbagen.2010.04.007
  » http://doi.org/10.1016/j.bbagen.2010.04.007
• LARSON, E.M., DOUGHMAN, D.J., GREGERSON, D.S. and OBRITSCH, W.F., 1997. A new, simple, nonradioactive, nontoxic in vitro assay to monitor corneal endothelial cell viability. Investigative Ophthalmology & Visual Science, vol. 38, no. 10, pp. 1929-1933. PMid:9331256.
• LIGASOVA, A. and KOBERNA, K., 2021. DNA dyes-highly sensitive reporters of cell quantification: comparison with other cell quantification methods. Molecules (Basel, Switzerland), vol. 26, no. 18, pp. 5515. http://doi.org/10.3390/molecules26185515 PMid:34576986.
  » http://doi.org/10.3390/molecules26185515
• MAHAR, J., SAEED, A., CHAUDHRY, G.S., IRFAN, M., CHANNAR, P.A., FAISAL, M. and LARIK, F.A., 2020. Synthesis, characterization and cytotoxic studies of novel 1,2,4-triazole-azomethine conjugates. Journal of the Indian Chemical Society, vol. 17, no. 4, pp. 943-951. http://doi.org/10.1007/s13738-019-01826-9
  » http://doi.org/10.1007/s13738-019-01826-9
• MAHTO, S.K., CHANDRA, P. and RHEE, S.W., 2010. In vitro models, endpoints and assessment methods for the measurement of cytotoxicity. Toxicology and Environmental Health Sciences, vol. 2, no. 2, pp. 87-93. http://doi.org/10.1007/BF03216487
  » http://doi.org/10.1007/BF03216487
• MAIER, K., SCHMITT-LANDGRAF, R. and SIEGEMUND, B., 1991. Development of an in vitro test system with human skin cells for evaluation of phototoxicity. Toxicology In Vitro, vol. 5, no. 5-6, pp. 457-461. http://doi.org/10.1016/0887-2333(91)90072-L PMid:20732056.
  » http://doi.org/10.1016/0887-2333(91)90072-L
• MASOOD, S., BABAR, U.M., CHAUDHRY, G.E., ASHFAQ, M., TAHIR, M.N. and ZAFAR, M.N., 2023. Zn (ii) and Cd (ii) pincer complexes bearing meta alkylated pyridinium amidates; synthesis & preliminary anticancer studies. New Journal of Chemistry, vol. 47, no. 47, pp. 21845-21853. http://doi.org/10.1039/D3NJ04131J
  » http://doi.org/10.1039/D3NJ04131J
• MASOOD, S., ZAFAR, M.N., CHAUDHRY, G.S., MUHAMMAD, T.S.T., DALEBROOK, A.F., NAZAR, M.F., MALIK, F.P., MUGHAL, E. and WRIGHT, L.J., 2019. Synthesis, characterization and anti-cancer properties of water-soluble bis (PYE) pro-ligands and derived palladium (II) complexes. Dalton Transactions (Cambridge, England), vol. 48, no. 41, pp. 15408-15418. http://doi.org/10.1039/C9DT01923E PMid:31393494.
  » http://doi.org/10.1039/C9DT01923E
• MEHMOOD, R., SADIQ, A., ALSANTALI, R.I., MUGHAL, E.U., ALSAHRIF, M.A., NAEEM, N., JAVID, A., AL-ROOQI, M.M., CHAUDHRY, G.E. and AHMED, S.A., 2022. Synthesis and evaluation of 1,3,5-Triaryl-2-pyrazoline derivatives as potent dual inhibitors of urease and α-glucosidase together with their cytotoxic, molecular modeling and drug-likeness studies. ACS Omega, vol. 7, no. 4, pp. 3775-3795. http://doi.org/10.1021/acsomega.1c06694 PMid:35128286.
  » http://doi.org/10.1021/acsomega.1c06694
• MILHERIO, A., NOZAKI, K., KLEVERLAAN, C.J., MURIS, J., MIURA, H. and FEILZER, A.J., 2016. In vitro cytotoxicity of metallic ions released from dental alloys. Odontology, vol. 104, no. 2, pp. 136-142. http://doi.org/10.1007/s10266-014-0192-z PMid:25549610.
  » http://doi.org/10.1007/s10266-014-0192-z
• MOFFA, E.B., IZUMIDA, F.E., MUSSI, M.C.M., SIQUEIRA, W.L., JORGE, J.H. and GIAMPAOLO, E.T., 2016. Interaction between XTT assay and candida albicans or streptococcus mutans viability. Journal of International Oral Health: JIOH, vol. 8, no. 1, pp. 12. http://doi.org/10.4103/0976-7428.199395
  » http://doi.org/10.4103/0976-7428.199395
• MOSMANN, T., 1983. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. Journal of Immunological Methods, vol. 65, no. 1-2, pp. 55-63. http://doi.org/10.1016/0022-1759(83)90303-4 PMid:6606682.
  » http://doi.org/10.1016/0022-1759(83)90303-4
• MOSS, B.J., KIM, Y., NANDAKUMAR, M.P. and MARTEN, M.R., 2008. Quantifying metabolic activity of filamentous fungi using a colorimetric XTT assay. Biotechnology Progress, vol. 24, no. 3, pp. 780-783. http://doi.org/10.1021/bp070334t PMid:18386938.
  » http://doi.org/10.1021/bp070334t
• NASEER, M.I., ZUBAIR, H. and KIM, M.O., 2009. Effect of fetal calf serum on cellular proliferation of mouse Y1 adrenocortical cells in vitro. Pakistan Journal of Medical Sciences, vol. 25, no. 3, pp. 500-504.
• NEMUDZIVHADI, V. and MASOKO, P., 2014. In vitro assessment of cytotoxicity, antioxidant, and anti-inflammatory activities of Ricinus communis (Euphorbiaceae) leaf extracts. Evidence-Based Complementary and Alternative Medicine, vol. 2014, no. 625961, pp. 625961. http://doi.org/10.1155/2014/625961 PMid:25477994.
  » http://doi.org/10.1155/2014/625961
• O’BRIEN, J., WILSON, I., ORTON, T. and POGNAN, F., 2000. Investigation of the alamar blue (resazurin) fluorescent dye for the assessment of mammalian cell cytotoxicity. European Journal of Biochemistry, vol. 267, no. 17, pp. 5421-5426. http://doi.org/10.1046/j.1432-1327.2000.01606.x PMid:10951200.
  » http://doi.org/10.1046/j.1432-1327.2000.01606.x
• PAGE, B., PAGE, M. and NOEL, C., 1993. A new fluorometric assay for cytotoxicity measurements in-vitro. International Journal of Oncology, vol. 3, no. 3, pp. 473-476. http://doi.org/10.3892/ijo.3.3.473 PMid:21573387.
  » http://doi.org/10.3892/ijo.3.3.473
• PRABST, K., ENGELHARDT, H., RINGGELER, S. and HUBNER, H., 2017. Basic colorimetric proliferation assays: MTT, WST, and Resazurin. Methods in Molecular Biology (Clifton, N.J.), vol. 1601, pp. 1-17. http://doi.org/10.1007/978-1-4939-6960-9_1 PMid:28470513.
  » http://doi.org/10.1007/978-1-4939-6960-9_1
• QUENT, V.M., LOESSNER, D., FRIIS, T., REICHERT, J.C. and HUTMACHER, D.W., 2010. Discrepancies between metabolic activity and DNA content as tool to assess cell proliferation in cancer research. Journal of Cellular and Molecular Medicine, vol. 14, no. 4, pp. 1003-1013. http://doi.org/10.1111/j.1582-4934.2010.01013.x PMid:20082656.
  » http://doi.org/10.1111/j.1582-4934.2010.01013.x
• ROEHM, N.W., RODGERS, G.H., HATFIELD, S.M. and GLASEBROOK, A.L., 1991. An improved colorimetric assay for cell proliferation and viability utilizing the tetrazolium salt XTT. Journal of Immunological Methods, vol. 142, no. 2, pp. 257-265. http://doi.org/10.1016/0022-1759(91)90114-U PMid:1919029.
  » http://doi.org/10.1016/0022-1759(91)90114-U
• SAHIN, B., 2023. In vitro cytotoxicity test methods: MTT and NRU. International Journal of PharmATA, vol. 3, no. 2, pp. 50-53. http://doi.org/10.5152/Pharmata.2023.1287964
  » http://doi.org/10.5152/Pharmata.2023.1287964
• SARI, C., KOLAYLI, S. and CELEP EYÜPOGLU, F., 2021. A comparative study of MTT and WST-1 assays in cytotoxicity analysis. Haydarpasa Numune Training and Research Hospital Medicinal Journal, vol. 61, pp. 281-288. http://doi.org/10.14744/hnhj.2019.16443
  » http://doi.org/10.14744/hnhj.2019.16443
• SCHIRMER, K., CHAN, A.G., GREENBERG, B.M., DIXON, D.G. and BOLS, N.C., 1997. Methodology for demonstrating and measuring the photocytotoxicity of fluoranthene to fish cells in culture. Toxicology In Vitro, vol. 11, no. 1-2, pp. 107-119. http://doi.org/10.1016/S0887-2333(97)00002-7 PMid:20654301.
  » http://doi.org/10.1016/S0887-2333(97)00002-7
• SCHREER, A., TINSON, C., SHERRY, J.P. and SCHIRMER, K., 2005. Application of Alamar blue/5-carboxyfluorescein diacetate acetoxymethyl ester as a noninvasive cell viability assay in primary hepatocytes from rainbow trout. Analytical Biochemistry, vol. 344, no. 1, pp. 76-85. http://doi.org/10.1016/j.ab.2005.06.009 PMid:16039980.
  » http://doi.org/10.1016/j.ab.2005.06.009
• SHAFIEE, M.A.M., ASRI, M.A.M. and ALWI, S.S.S., 2021. Review on the in vitro cytotoxicity assessment in accordance to the International Organization for Standardization (ISO). Malaysian Journal of Medicine & Health Sciences, vol. 17, no. 2, pp. 261-269.
• SKEHAN, P., STORENG, R., SCUDIERO, D., MONKS, A., MCMAHON, J., VISTICA, D., WARREN, J.T., BOKESCH, H., KENNY, S. and BOYD, M.R., 1990. New colorimetric cytotoxicity assay for anticancer-drug screening. Journal of the National Cancer Institute, vol. 82, no. 13, pp. 1107-1112. http://doi.org/10.1093/jnci/82.13.1107 PMid:2359136.
  » http://doi.org/10.1093/jnci/82.13.1107
• STODDART, M.J., 2011. Cell viability assays: introduction. Methods in Molecular Biology (Clifton, N.J.), vol. 740, pp. 1-6. http://doi.org/10.1007/978-1-61779-108-6_1 PMid:21468961.
  » http://doi.org/10.1007/978-1-61779-108-6_1
• STROBER, W., 2001. Trypan blue exclusion test of cell viability. Current Protocols in Immunology, vol. 3, no. Appendix 3, pp. 3B. http://doi.org/10.1002/0471142735.ima03bs21 PMid:18432654.
  » http://doi.org/10.1002/0471142735.ima03bs21
• SUMANTRAN, V.N., 2011. Cellular chemosensitivity assays: an overview. Methods in Molecular Biology (Clifton, N.J.), vol. 731, pp. 219-236. http://doi.org/10.1007/978-1-61779-080-5_19 PMid:21516411.
  » http://doi.org/10.1007/978-1-61779-080-5_19
• SYLVESTER, P.W., 2011. Optimization of the tetrazolium dye (MTT) colorimetric assay for cellular growth and viability. Methods in Molecular Biology (Clifton, N.J.), vol. 716, pp. 157-168. http://doi.org/10.1007/978-1-61779-012-6_9 PMid:21318905.
  » http://doi.org/10.1007/978-1-61779-012-6_9
• THOMAS, M., FINNEGAN, C.E., ROGERS, K.M.A., PURCELL, J.W., TRIMBLE, A., JOHNSTON, P.G. and BOLAND, M.P., 2004. STAT1: A modulator of chemotherapy-induced apoptosis. Cancer Research, vol. 64, no. 22, pp. 8357-8364. http://doi.org/10.1158/0008-5472.CAN-04-1864 PMid:15548705.
  » http://doi.org/10.1158/0008-5472.CAN-04-1864
• TRAN, S.L., PUHAR, A., NGO-CAMUS, M. and RAMARAO, N., 2011. Trypan blue dye enters viable cells incubated with the pore-forming toxin HlyII of Bacillus cereus. PLoS One, vol. 6, no. 9, pp. e22876. http://doi.org/10.1371/journal.pone.0022876 PMid:21909398.
  » http://doi.org/10.1371/journal.pone.0022876
• UDE, A., AFI-LESLIE, K., OKEKE, K. and OGBODO, E., 2022. Trypan blue exclusion assay, neutral red, acridine orange and propidium iodide. In: S. ANIL and M.A. MANSOUR, eds. Cytotoxicity: understanding cellular damage and response Londres: IntechOpen, pp. 1-12.
• VEGA-AVILA, E. and PUGSLEY, M.K., 2011. An overview of colorimetric assay methods used to assess survival or proliferation of mammalian cells. Proceedings of the Western Pharmacology Society, vol. 54, pp. 10-14. PMid:22423572.
• VICHAI, V. and KIRTIKARA, K., 2006. Sulforhodamine B colorimetric assay for cytotoxicity screening. Nature Protocols, vol. 1, no. 3, pp. 1112-1116. http://doi.org/10.1038/nprot.2006.179 PMid:17406391.
  » http://doi.org/10.1038/nprot.2006.179
• VINKEN, M. and BLAAUBOER, B.J., 2017. In vitro testing of basal cytotoxicity: establishment of an adverse outcome pathway from chemical insult to cell death. Toxicology In Vitro, vol. 39, pp. 104-110. http://doi.org/10.1016/j.tiv.2016.12.004 PMid:27939612.
  » http://doi.org/10.1016/j.tiv.2016.12.004
• WEYERMANN, J., LOCHMANN, D. and ZIMMER, A., 2005. A practical note on the use of cytotoxicity assays. International Journal of Pharmaceutics, vol. 288, no. 2, pp. 369-376. http://doi.org/10.1016/j.ijpharm.2004.09.018 PMid:15620877.
  » http://doi.org/10.1016/j.ijpharm.2004.09.018
• YUNUS, U., KHAN, M.E., SADIQ, S., AAMIR, M., ULLAH, Z., BHATTI, M.H., SHER, M. and CHAUDHRY, G.-S., 2024. Methotrexate-loaded Fe-metal organic frameworks: Synthesis, characterizations, and drug release investigations. Journal of Drug Delivery Science and Technology, vol. 97, pp. 105790. http://doi.org/10.1016/j.jddst.2024.105790
  » http://doi.org/10.1016/j.jddst.2024.105790
• YUNUS, U., ZULFIQAR, M.A., AJMAL, M., BHATTI, M.H., CHAUDHRY, G.E., MUHAMMAD, T.S.T. and SUNG, Y.Y., 2020. Targeted drug delivery systems: synthesis and in vitro bioactivity and apoptosis studies of gemcitabine-carbon dot conjugates. Biomedical Materials (Bristol, England), vol. 15, no. 6, pp. 065004. http://doi.org/10.1088/1748-605X/ab95e1 PMid:32442994.
  » http://doi.org/10.1088/1748-605X/ab95e1
• ZACHARI, M.A., CHONDROU, P.S., POULILIOU, S.E., MITRAKAS, A.G., ABATZOGLOU, I., ZOIS, C.E. and KOUOURAKIS, M.I., 2013. Evaluation of the alamarblue assay for adherent cell irradiation experiments. Dose-Response, vol. 12, no. 2, pp. 246-258. http://doi.org/10.2203/dose-response.13-024.Koukourakis PMid:24910583.
  » http://doi.org/10.2203/dose-response.13-024.Koukourakis
• ZIVADINOVIC, D., GAMETCHU, B. and WATSON, C.S., 2005. Membrane estrogen receptor-alpha levels in MCF-7 breast cancer cells predict cAMP and proliferation responses. Breast Cancer Research, vol. 7, no. 1, pp. 101-112. http://doi.org/10.1186/bcr958 PMid:15642158.
  » http://doi.org/10.1186/bcr958

Publication Dates

• Publication in this collection
  13 Dec 2024
• Date of issue
  2024

History

• Received
  12 Mar 2024
• Accepted
  19 Sept 2024