Cytotoxicity assays were performed to determine functionality of labelled NK cells and retargeted genetically modified NK cells expressing chimeric. Cytotoxicity assays are widely used in fundamental research and in drug discovery to screen libraries for toxic compounds. A compound generating a cytotoxic. Cell Proliferation and Cytotoxicity Assays. Adan A, Kiraz Y, Baran Y(1). Author information: (1)İzmir Institute of Technology, Department of Molecular Biology and.
In vitro, chemicals such as drugs and pesticides have different cytotoxicity mechanisms such as destruction of cell membranes, prevention of protein synthesis, irreversible binding to receptors etc. In order to determine the cell death caused by these damages, there is a need for cheap, reliable and reproducible short-term cytotoxicity and cell viability assays.
Cytotoxicity and cell viability assays are based on various cell functions. A broad spectrum of cytotoxicity assays is currently used in the fields of toxicology and pharmacology. There are different classifications for these assays: Choosing the appropriate method among these assays is important for obtaining accurate and reliable results. When selecting the cytotoxicity and cell viability assays to be used in the study, different parameters have to be considered such as the availability in the laboratory where the study is to be performed, test compounds, detection mechanism, specificity, and sensitivity.
The aim of this chapter is to guide the researcher interested in this subject to select the appropriate assay for their study.
Physical and chemical agents can affect cell health and metabolism. These agents may cause toxicity on cells via different mechanisms such as destruction of cell membranes, prevention of protein synthesis, irreversible binding to receptors, inhibition of polydeoxynucleotide elongation, and enzymatic reactions [ 1 ].
In order to determine the cell death caused by these mechanisms, there is a need for cheap, reliable and reproducible short-term cytotoxicity and cell viability assays. In vitro cell viability and cytotoxicity assays with cultured cells are widely used for cytotoxicity tests of chemicals and for drug screening.
Application of these assays has been of increasing interest over recent years. Currently, these assays are also used in oncological researches to evaluate both compound toxicity and tumor cell growth inhibition during drug development. Because, they are rapid, inexpensive and do not require the use of animals. Furthermore, they are useful for testing large number of samples. Cell viability and cytotoxicity assays are based on various cell functions such as cell membrane permeability, enzyme activity, cell adherence, ATP production, co-enzyme production, and nucleotide uptake activity [ 1 ].
However, they have some disadvantages because they are not technically advanced enough yet, to replace animal tests [ 2 ]. A broad spectrum of cytotoxicity and cell viability assays is currently used in the fields of toxicology and pharmacology.
The choice of assay method is crucial in the assessment of the interaction type [ 3 ]. Although there are different classifications for cytotoxicity and cell viability assays, in this chapter, these assays are classified according to measurement types of end points color changes, fluorescence, luminescent etc. The proportion of viable cells in a cell population can be estimated in various methods. The simplest and widely used one of the methods is dye exclusion method.
In dye exclusion method, viable cells exclude dyes, but dead cells not exclude them. Although the staining procedure is quite simple, experimental procedure of large number of samples is difficult and time consuming [ 4 ]. Determination of membrane integrity is possible via dye exclusion method. A variety of such dyes have been employed, including eosin, Congo red, erythrosine B, and trypan blue [ 5 , 6 ].
Of the dyes listed, trypan blue has been used the most extensively [ 7 , 8 , 9 , 10 ]. If dye exclusion assays are used, following factors must be considered i lethally damaged cells by cytotoxic agents may require several days to lose their membrane integrity, ii the surviving cells may continue to proliferate during this time, and iii some lethally damaged cells are not appear to be stained with dye at the end of the culture period, because they may undergo an early disintegration. Factors ii and iii may cause an underestimate of cell death when the results of the assay are based on percent viability expression [ 11 , 12 , 13 ].
Dye exclusion assays have unique advantages for chemosensitivity testing. They are comparatively simple, require small numbers of cells, are rapid, and are capable of detecting cell kill in nondividing cell populations. Further investigations into the possible role of these assays in chemosensitivity testing are warranted [ 11 ]. However, none of these dyes is recommended for use on monolayer cell cultures but rather they are intended for cells in suspension; thus monolayer cells must first trypsinized [ 6 ].
Trypan blue is a large negatively charged molecule. Trypan blue dye exclusion assay is based on the principle that live cells possess intact cell membranes that exclude this dye, whereas dead cells do not. In this assay, adherent or nonadherent cells are incubated with serial dilutions of test compounds for various times. After the compound treatment, cells are washed and suspended.
Cell suspension is mixed with dye and then visually examined to determine whether cells take up or exclude dye. Viable cells will have a clear cytoplasm, whereas dead cells will have a blue cytoplasm [ 14 , 15 ]. This method is simple, inexpensive, and a good indicator of membrane integrity [ 17 ], and dead cells are colored blue within seconds of exposure to the dye [ 18 ].
Cell counting is generally done using a hemacytometer [ 19 ]. Counting errors have been attributed to poor dispersion of cells, cell loss during cell dispersion, inaccurate dilution of cells, improper filling of the chamber and presence of air bubbles in the chamber [ 17 ].
While the staining procedure is quite simple, it is difficult to process large number of samples concurrently, particularly where the exact timing of progressive cytotoxic effects is required [ 4 ]. Furthermore, trypan blue staining cannot be used to distinguish between the healthy cells and the cells that are alive but losing cell functions. Another disadvantage of trypan blue is toxic side effect of this dye on mammalian cells [ 20 ].
Erythrosine B, also known as erythrosine or Red No. Erythrosine B has already been introduced as a vital dye for counting viable cells. Principle of this dye exclusion assay is similar to trypan blue dye exclusion assay principle.
Although erythrosine B is an alternative bio-safe vital dye for cell counting; it is not widely used to count viable or dead cells. It has benefits such as low cost, versatility, and bio-safety [ 20 ].
Its procedure is time-consuming and labor-intensive. Moreover, potential disadvantages include contamination of reusable cell counting chamber, variations of hemocytometer filling rates, and inter-user variations [ 20 ]. Principle of colorimetric assays is the measurement of a biochemical marker to evaluate metabolic activity of the cells. Reagents used in colorimetric assays develop a color in response to the viability of cells, allowing the colorimetric measurement of cell viability via spectrophotometer.
Colorimetric assays are applicable for adherent or suspended cell lines, easy to perform, and comparably economical [ 22 , 23 ]. Commercial kits of colorimetric assays are available from several companies and generally experimental procedures of these assays are available in kit packages. MTT 3- 4,5-dimethylthiazolyl -2—5-diphenyltetrazolium bromide assay is one of the most commonly used colorimeteric assay to assess cytotoxicity or cell viability [ 24 ]. This assay determines principally cell viability through determination of mitochondrial function of cells by measuring activity of mitochondrial enzymes such as succinate dehydrogenase [ 18 ].
This product can be quantified by light absorbance at a specific wavelength. This method is far superior to the previously mentioned dye exclusion methods because it is easy to use, safe, has a high reproducibility, and is widely used to determine both cell viability and cytotoxicity tests [ 18 , 25 ]. MTT formazan is insoluble in water, and it forms purple needle-shaped crystals in the cells. Therefore, prior to measuring the absorbance, an organic solvent such as dimethyl sulfoxide DMSO or isopropanol is required to solubilize the crystals.
Additionally, the cytotoxicity of MTT formazan makes it difficult to remove cell culture media from the plate wells due to floating cells with MTT formazan needles, giving significant well-to-well error [ 18 , 26 ].
Additional control experiments should be conducted to reduce false-positive or false-negative results that caused by background interference due to inclusion of particles.
This interference could lead to an overestimation of the cell viability. This can often be controlled by subtraction of the background absorbance of the cells in the presence of the particles, but without the assay reagents [ 18 , 26 ].
The MTS assay 5- 3-carboxymethoxyphenyl 4,5-dimethyl-thiazoly 4-sulfophenyl tetrazolium, inner salt assay is a colorimetric assay. This assay is based on the conversion of a tetrazolium salt into a colored formazan by mitochondrial activity of living cells. Performance of this assay is very competitive to other toxicological tests. This assay provides ideal properties for cytotoxicity measurement because it is easy to use, rapid, reliable, and inexpensive. Therefore, it can be used for onsite toxicological assessments [ 27 , 29 , 30 , 31 ].
The proportion of MTS detection reagents to cells in culture also influences the measured absorbance level. While MTT produced a water-insoluble formazan compound which required dissolving the dye in order to measure its absorbance, the XTT produces a water-soluble dye.
The procedure of XTT is simply for measuring proliferation and is therefore an excellent solution for quantitating cells and determining their viability. XTT is used to assay cell proliferation as response to different growth factors. It is also used for assaying cytotoxicity. This assay is based on the ability reduction of the tetrazolium salt XTT to orange-colored formazan compounds by metabolic active cells. Orange-colored formazan is water soluble and its intensity can be measured with a spectrophotometer.
There is a linear relationship between the intensity of the formazan and the number of viable cells. The use of multiwell plates and a spectrophotometer or ELISA reader allows for study with a large number of samples and obtaining results easily and rapidly. During the incubation time, an orange color is formed and the intensity of color can be measured with a spectrophotometer [ 34 , 35 ].
XTT assay is speed, sensitive, easy to use, and safe method. It has high sensitivity and accuracy [ 35 ]. XTT assay performance depends on reductive capacity of viable cells with the mitochondrial dehydrogenase activity. Therefore, changes of reductive capacity of viable cells resulting from enzymatic regulation, pH, cellular ion concentration e. WST-1 2- 4-iodophenyl 4-nitrophenyl 2,4-disulfophenyl -2 H tetrazolium monosodium salt cell proliferation assay is a simple, colorimetric assay designed to measure the relative proliferation rates of cells in culture.
The principle of this assay is based on the conversion of the tetrazolium salt WST-1 into a highly water-soluble formazan by mitochondrial dehydrogenase enzymes in the presence of intermediate electron acceptor, such as mPMS 1-methoxymethyl-phenazinium methyl sulfate [ 36 ].
The water-soluble salt is released into the cell culture medium. Within incubation period, the reaction produces a color change which is directly proportional to the amount of mitochondrial dehydrogenase in cell culture and thus, the assay measures the metabolic activity of cells.
To perform the assay, the WST-1 reagent that is ready-to-use is added directly into the media of cells cultured in multiwell plates. It is easy to use, safe, has a high reproducibility, and is widely used to determine both cell viability and cytotoxicity tests. Furthermore, phenol red indicators in cell culture medium do not interfere with the dye reaction.
Because the colored dye which produced at the end of experiment is water-soluble, it is not required a solvent and additional incubation time [ 37 ]. Whether one-time addition of WST-1 can reflect the effect of the testing agents at different time points on the trend of relative cell viability is still unclear [ 37 ]. WST-8 assay is a colorimetric assay for the determination of viable cell numbers and can be used for cell proliferation assays as well as cytotoxicity assays.
It is more sensitive than WST-1 particularly at neutral pH [ 37 ]. Since WST-8, WST-8 formazan, and 1-methoxy PMS have no cytotoxicity on cells in the culture media, same cells from the previous assay may be used for additional experiments.
WST-8 is not cell permeable, which results in low cytotoxicity. Therefore, after the assay, it is possible to continue further experiments using the same cells. Furthermore, it produces the water-soluble formazan upon cellular reduction, which would provide an additional advantage to the method by allowing a simpler assay procedure and not required an extra step to dissolve the formazan [ 28 ].
An important consideration is that reduction of assay substrates is impacted by changes in intracellular metabolic activity that has no direct effect on overall cell viability [ 15 ]. LDH lactate dehydrogenase cytotoxicity assay is a colorimetric method of assaying cellular cytotoxicity. LDH Cytotoxicity Assay Kit can be used with different cell types not only for assaying cell-mediated cytotoxicity but also for assessment of cytotoxicity mediated by toxic chemicals and other test compounds.
The assay measures the stable, cytosolic, lactate dehydrogenase LDH enzyme quantitatively. This enzyme releases from damaged cells.
LDH is an enzyme that is normally found within the cell cytoplasm. When cell viability reduced leakiness of the plasma membrane increase and therefore LDH enzyme is released into the cell culture medium. The released LDH is measured with a coupled enzymatic reaction that results in the conversion of a tetrazolium salt iodonitrotetrazolium INT into a red color formazan by diaphorase.
In addition, well-known membranolytic particles such as crystalline silica can be used as a positive control in LDH assay [ 40 ]. Reliability, speed, and simple evaluation are some of characteristics of this assay. Because, the loss of intracellular LDH and its release into the culture medium is an indicator of irreversible cell death due to cell membrane damage [ 38 , 41 ].
The major limitation of this assay is that serum and some other compounds have inherent LDH activity. For example, the fetal calf serum has extremely high background readings. At a minimum, you should always first test the assay with an unused aliquot of the media you intend to use and compare the reading to that from media lacking supplements e. SRB Sulforhodamine B assay is a rapid and sensitive colorimetric method for measuring the drug-induced cytotoxicity in both attached and suspension cell cultures.
This assay as first described by Skehan and colleagues was developed for use in the disease-orientated, large-scale anticancer drug discovery program of the National Cancer Institute NCI that was launched in SRB is a bright pink aminoxanthene dye with two sulfonic groups.
Under mildly acidic conditions, SRB binds to protein basic amino acid residues in TCA-fixed trichloroacetic acid cells to provide a sensitive index of cellular protein.
SRB assay is also used to evaluate colony formation and colony extinction [ 43 ]. The SRB assay is simple, fast, and sensitive. It provided good linearity with cell number, permitted the use of saturating dye concentrations, is less sensitive to environmental fluctuations, is independent of intermediary metabolism, and provided a fixed end point that is not require a time-sensitive measurement of initial reaction velocity [ 43 ].
Reproducibility of this assay is high. It is important to obtain and maintain a homogeneous cell suspension. This assay was developed by Borenfreund and Puerner [ 44 ]. This assay was based on the ability of viable cells to take up the supravital dye neutral red. You have 2 remaining attempts. You have 1 remaining attempt. This field is required. There was an issue with the password reset process. Please try again or contact Customer Service.
Log in with Your New Password. You have not verified your email address. A verified email address is required to access the full functionality of your Promega. Our customer and technical support experts are available by telephone, fax, email or live chat to help. We offer an extensive line of effective and innovative assays and reagents for determining cell viability and cytotoxicity. Choose from assays to measure viability in cell culture, 3D microtissues, bacterial cultures and virus-infected cells.
Many can be multiplexed with apoptosis and other viability assays to determine mechanisms of cell death and sensitively compare data from well-to well, plate-to-plate, and day-to-day. Sensitive detection of cytotoxicity for samples low in cell number, including 3D microtissue cultures.
A bioluminescent method to kinetically monitor viability in cell culture up to 72 hours. A homogeneous method optimized to assess viability in 3D cell culture. A quantifiable method of determining viral-induced cytopathic effects CPE in host cells caused by lytic virions. Detergent solution useful for lysing cells and creating a cytotoxicity positive control. An assay to kinetically monitor cytotoxicity up to 72 hours with multiplex capability.
Gold standard method for determining cell viability based on quantitation of ATP. Detergent solution for permeabilizing cells and creating a cytotoxicity chemistry positive control.
Highly sensitive luminescent cytotoxicity assay that measures the relative number of dead cells. A fluorescent and luminescent assay that measures the relative number of live and dead cells in a population. A single-reagent-addition, homogeneous, fluorescent assay that measures the relative number of dead cells in cell populations.
A single-reagent-addition fluorescent assay that measures the number of live and dead cells in culture wells. Measure the number of viable microbial cells in culture with a luminescent signal proportional to the amount of ATP present. Monitor cell viability with this homogeneous, resazurin, fluorescent assay. Showing 25 of 25 Products. Our website does not fully support your browser. Your Account Username Account not found.
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Assays to measure proliferation, viability and cytotoxicity are commonly used to monitor the response and health of cells in culture after treatment with various. We offer an extensive line of effective and innovative assays and reagents for determining cell viability and cytotoxicity. Choose from assays to measure viability. Cytotoxicity is the quality of being toxic to cells. Examples of toxic agents are an immune cell or Cytotoxicity assays are widely used by the pharmaceutical industry to screen for cytotoxicity in compound libraries. Researchers can either look.