Trypan Blue Exclusion Assay, Neutral Red, Acridine Orange and Propidium Iodide (original) (raw)
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Cell Viability Measurement - Trypan Blue versus Acridine Orange-Propidium Iodide
A reliable method for rapidly determining the viability of HPCs is essential for clinical cellular engineering. The trypan blue (TB) viability assay is a widely performed procedure to assess HPC viability before and after cryopreservation and before bone marrow transfusion. 1 The TB assay is based on the ability of the membrane of viable cells to exclude the dye, while nonviable cells are stained blue when viewed by light microscopy. However, the TB assay can be difficult to interpret because of staining artifacts. In addition, the TB viability assay has never been validated for use with HPCs.
Mutation Research/Environmental Mutagenesis and Related Subjects, 1993
Rat hepatocytes treated in vitro with A2RA, an angiotensin II receptor antagonist, displayed an increased level of DNA-strand breaks as determined by alkaline elution, without an appreciable increase in cytotoxicity as determined by a trypan blue dye exclusion assay at harvest. The alkaline elution profile appeared to have two components: a rapidly eluting component detected in the first fraction collected (often associated with DNA from dead or dying cells), followed by a more slowly eluting component detected in the subsequent fractions. Further analysis of hepatocytes treated with A2RA by pulsed-field gel electrophoresis and neutral elution revealed significant levels of DNA double-strand breaks. Electron microscopy (EM) showed pronounced damage to mitochondria; although cell blebbing was seen using both EM and light microscopy, the plasma and nuclear membranes appeared intact when examined by EM. Cellular ATP levels decreased precipitously with increasing doses of A2RA, falling to less than 10% of control values at a dose of 0.213 mM A2RA, a concentration showing 100% relative viability by trypan blue at harvest. Thus, whereas in our experience trypan blue dye exclusion accurately reflects cytotoxicity induced by the majority of test agents, in this rather unusual case, trypan blue did not accurately reflect compound-induced cytotoxicity at harvest since there was no concurrent loss of membrane integrity. However, when hepatocytes treated with A2RA were incubated for either 3 h or 20 h in the absence of compound, a sharp, dose-dependent decline in viability was observed using trypan blue dye exclusion. Together with the initial, dose-dependent drop in the alkaline elution curve, these data suggest that the observed DNA double-strand breaks arose as a consequence of endonucleolytic DNA degradation associated with cytotoxicity, rather than by a direct compound-DNA interaction. Since DNA doublestrand breaks behave under alkaline denaturing conditions as two single-strand breaks and can therefore produce increases in the alkaline-elution slope values, a necessary criteria for a valid positive result in
Colorimetric Cytotoxicity Assays
IntechOpen eBooks, 2022
Cytotoxicity experiments are carried out to evaluate whether a chemical has cytotoxic potential. Because of its ease of use and compatibility with data collected from in vivo investigations, cell-based cytotoxicity studies have emerged as a viable alternative to animal trials in research. Cell-damaging events such as apoptosis, autophagy, and necrosis may occur after exposure to cytotoxic substances. Thanks to the cell-based cytotoxicity studies, basic information is obtained about the cytotoxic effects of the tested substance. To measure cell viability, a variety of techniques are used. Regardless of the sort of cytotoxicity investigation that was carried out, the crucial thing is to figure out how much metabolic activity there is in the cells at the end of the experiment. Cytotoxicity detection methods are generally colorimetric, luminescent, and enzymatic methods. In colorimetric methods, measurement is based on color change using tetrazolium salts, such as MTT, MTS, XTT, WST. Three main steps are followed in tetrazolium compound toxicity tests. Toxic compounds are introduced to cells in the initial stage. The poisonous chemical is eliminated in the second phase and followed by the addition of the tetrazolium compound. The metabolically active cells are determined in the last stage by using a spectrophotometric approach to measure color change.
Improved sensitivity of trypan blue dye exclusion assay with Ni2+ or Co2+ salts
Cytotechnology, 2000
A modified trypan blue dye exclusion assay was developed usingNi(2+) or Co(2+) salts to determine the viability ofprimary and transformed cells. When the cells were preincubatedwith NiSO(4) or CoCl(2) followed by trypan blue assay, thecontrast between stained and unstained cells was significantlyincreased as compared to the conventional trypan blue dyeexclusion assay.
HPC viability measurement: trypan blue versus acridine orange and propidium iodide
Transfusion, 2000
A reliable method for rapidly determining the viability of HPCs is essential for clinical cellular engineering. The trypan blue (TB) viability assay is a widely performed procedure to assess HPC viability before and after cryopreservation and before bone marrow transfusion. 1 The TB assay is based on the ability of the membrane of viable cells to exclude the dye, while nonviable cells are stained blue when viewed by light microscopy. However, the TB assay can be difficult to interpret because of staining artifacts. In addition, the TB viability assay has never been validated for use with HPCs.
Cancer Research
The following factors must be considered when dye exclu sion assays are interpreted, (a) It may require several days for lethally damaged cells to lose their membrane integrity follow ing a cytotoxic insult, (b) During this time, the "surviving" cells may continue to proliferate, (c) Also during this time, some lethally damaged cells may undergo an early disintegration, so that they are not present to be stained with dye at the end of the culture period. Factors b and c may cause an underestimate of cell kill when the results of the assay are based upon the traditional "percent viability" expression. In order to overcome these problems, an internal standard was developed and tested. This was based upon the addition of a constant number of permanently fixed duck erythrocytes to the cultures of cells from two different established tumor cell lines. Results were based upon comparisons of the ratios of "viable" tumor cells to duck erythrocytes on permanent cytocentrifuge slides pre pared from the cultures. This novel "ratio" method was found to be a more sensitive index of drug-induced cell kill than the traditional percent viability method. A standard agar cloning assay gave somewhat higher estimates of cell kill than the ratio method, although both assays were in qualitative agreement for the drugs tested. All three assays demonstrated a clear dose-effect relationship for most of the drugs tested. Dye exclusion assays may have a useful role in chemosensitivity testing in vitro.
In Vitro Cytotoxicity and Cell Viability Assays: Principles, Advantages, and Disadvantages
Genotoxicity - A Predictable Risk to Our Actual World
Cytotoxicity is one of the most important indicators for biological evaluation in vitro studies. 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: (i) dye exclusion assays; (ii) colorimetric assays; (iii) fluorometric assays; and (iv) luminometric 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. In this chapter, information will be given about in vitro cytotoxicity and viability assays, these assays will be classified and their advantages and disadvantages will be emphasized. The aim of this chapter is to guide the researcher interested in this subject to select the appropriate assay for their study.
Trypan blue exclusion assay by flow cytometry
Brazilian Journal of Medical and Biological Research, 2014
Dye exclusion tests are used to determine the number of live and dead cells. These assays are based on the principle that intact plasma membranes in live cells exclude specific dyes, whereas dead cells do not. Although widely used, the trypan blue (TB) exclusion assay has limitations. The dye can be incorporated by live cells after a short exposure time, and personal reliability, related to the expertise of the analyst, can affect the results. We propose an alternative assay for evaluating cell viability that combines the TB exclusion test and the high sensitivity of the flow cytometry technique. Previous studies have demonstrated the ability of TB to emit fluorescence when complexed with proteins. According to our results, TB/bovine serum albumin and TB/cytoplasmic protein complexes emit fluorescence at 660 nm, which is detectable by flow cytometry using a 650-nm low-pass band filter. TB at 0.002% (w/v) was defined as the optimum concentration for distinguishing unstained living cells from fluorescent dead cells, and fluorescence emission was stable for 30 min after cell treatment. Although previous studies have shown that TB promotes green fluorescence quenching, TB at 0.002% did not interfere with green fluorescence in human live T-cells stained with anti-CD3/fluorescein isothiocyanate (FITC) monoclonal antibody. We observed a high correlation between the percentage of propidium iodide+ +CD3/FITC + and TB+ +CD3/FITC + cells, as well as similar doublestained cell profiles in flow cytometry dot-plot graphs. Taken together, the results indicate that a TB exclusion assay by flow cytometry can be employed as an alternative tool for quick and reliable cell viability analysis.
Proceedings of the Western Pharmacology Society, 2011
The aim of this review is to briefly describe some colorimetric methods that are commonly used to evaluate a new chemical entity (NCE) on cell cultures in non-clinical oncology discovery research. These methods have the distinct advantage over other techniques in that they can be applied and used in a cell monolayer or a suspension culture. Both protein assay determination and cell viability assays may be conducted using these culture systems. The viability of cell cultures is routinely assessed by utilizing the metabolic capacity of cells which biochemically convert chemicals (usually color dyes) which can then be conveniently measured at specific wavelengths using a multi-well plate reader. Resazurin (Alamar Blue) is an example of one of these metabolically active compounds. Resazurin is a nontoxic dye that can also be used to measure migration and cellular invasion without resorting to sacrifice of the cells during the test procedure. Another is 5-bromo-2-deoxyuridine (bromodeoxy...