Cell Viability Assays

doi:10.1007/978-1-4939-6960-9

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Cell Viability Assays

Book•DOI•

Cell Viability Assays

01 Jan 2017-

TL;DR: This chapter describes selected assays for the evaluation of cellular viability and proliferation of cell cultures using the formation of the omnipresent reducing agents NADH and NADPH as a marker for metabolic activity in the following assays.

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Abstract: This chapter describes selected assays for the evaluation of cellular viability and proliferation of cell cultures. The underlying principle of these assays is the measurement of a biochemical marker to evaluate the cell’s metabolic activity. The formation of the omnipresent reducing agents NADH and NADPH is used as a marker for metabolic activity in the following assays. Using NADH and NADPH as electron sources, specific dyes are biochemically reduced which results in a color change that can be determined with basic photometrical methods. The assays selected for this chapter include MTT, WST, and resazurin. They are applicable for adherent or suspended cell lines, easy to perform, and comparably economical. Detailed protocols and notes for easier handling and avoiding pitfalls are enclosed to each assay.

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Journal Article•DOI•

SARS-coronavirus-2 replication in Vero E6 cells: replication kinetics, rapid adaptation and cytopathology.

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Natacha S. Ogando¹, Tim J. Dalebout¹, Jessika C. Zevenhoven-Dobbe¹, Ronald W. A. L. Limpens¹, Yvonne van der Meer¹, Leon Caly², Julian Druce², Jutte J.C. de Vries¹, Marjolein Kikkert¹, Montserrat Bárcena¹, Igor A. Sidorov¹, Eric J. Snijder¹ - Show less +8 more•Institutions (2)

Leiden University Medical Center¹, Royal Melbourne Hospital²

01 Sep 2020-Journal of General Virology

TL;DR: The sensitivity of the two viruses to three established inhibitors of coronavirus replication is very similar, but that SARS-CoV-2 infection was substantially more sensitive to pre-treatment of cells with pegylated interferon alpha.

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Abstract: The sudden emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) at the end of 2019 from the Chinese province of Hubei and its subsequent pandemic spread highlight the importance of understanding the full molecular details of coronavirus infection and pathogenesis Here, we compared a variety of replication features of SARS-CoV-2 and SARS-CoV and analysed the cytopathology caused by the two closely related viruses in the commonly used Vero E6 cell line Compared to SARS-CoV, SARS-CoV-2 generated higher levels of intracellular viral RNA, but strikingly about 50-fold less infectious viral progeny was recovered from the culture medium Immunofluorescence microscopy of SARS-CoV-2-infected cells established extensive cross-reactivity of antisera previously raised against a variety of non-structural proteins, membrane and nucleocapsid protein of SARS-CoV Electron microscopy revealed that the ultrastructural changes induced by the two SARS viruses are very similar and occur within comparable time frames after infection Furthermore, we determined that the sensitivity of the two viruses to three established inhibitors of coronavirus replication (remdesivir, alisporivir and chloroquine) is very similar, but that SARS-CoV-2 infection was substantially more sensitive to pre-treatment of cells with pegylated interferon alpha An important difference between the two viruses is the fact that - upon passaging in Vero E6 cells - SARS-CoV-2 apparently is under strong selection pressure to acquire adaptive mutations in its spike protein gene These mutations change or delete a putative furin-like cleavage site in the region connecting the S1 and S2 domains and result in a very prominent phenotypic change in plaque assays

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445 citations

Cites methods from "Cell Viability Assays"

...After 3 days, a colorimetric cell viability assay [60] was used to measure drug toxicity and inhibition of virus replication in mock- and virusinfected cells, respectively....
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Journal Article•DOI•

Consensus guidelines for the use and interpretation of angiogenesis assays

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Patrycja Nowak-Sliwinska¹, Kari Alitalo², Elizabeth Allen³, Andrey Anisimov², Alfred C. Aplin⁴, Robert Auerbach⁵, Hellmut G. Augustin⁶, Hellmut G. Augustin⁷, David O. Bates⁸, Judy R. van Beijnum⁹, R. Hugh F. Bender¹⁰, Gabriele Bergers³, Gabriele Bergers¹¹, Andreas Bikfalvi¹², Joyce Bischoff¹³, Barbara C. Böck⁶, Barbara C. Böck⁷, Peter C. Brooks¹⁴, Federico Bussolino¹⁵, Bertan Cakir¹³, Peter Carmeliet³, Daniel Castranova¹⁶, Anca Maria Cimpean, Ondine Cleaver¹⁷, George Coukos¹⁸, George E. Davis¹⁹, Michele De Palma²⁰, Anna Dimberg²¹, Ruud P.M. Dings²², Valentin Djonov²³, Andrew C. Dudley²⁴, Neil Dufton²⁵, Sarah-Maria Fendt³, Napoleone Ferrara²⁶, Marcus Fruttiger²⁷, Dai Fukumura¹³, Bart Ghesquière³, Bart Ghesquière²⁸, Yan Gong¹³, Robert J. Griffin²², Adrian L. Harris²⁹, Christopher C.W. Hughes¹⁰, Nan W. Hultgren¹⁰, M. Luisa Iruela-Arispe³⁰, Melita Irving¹⁸, Rakesh K. Jain¹³, Raghu Kalluri³¹, Joanna Kalucka³, Robert S. Kerbel³², Jan Kitajewski³³, Ingeborg Klaassen³⁴, Hynda K. Kleinmann³⁵, Pieter Koolwijk¹⁸, Elisabeth Kuczynski³², Brenda R. Kwak¹, Koen Marien, Juan M. Melero-Martin¹³, Lance L. Munn¹³, Roberto F. Nicosia⁴, Agnès Noël³⁶, Jussi Nurro³⁷, Anna-Karin Olsson²¹, Tatiana V. Petrova³⁸, Kristian Pietras, Roberto Pili³⁹, Jeffrey W. Pollard⁴⁰, Mark J. Post⁴¹, Paul H.A. Quax⁴², Gabriel A. Rabinovich⁴³, Marius Raica, Anna M. Randi²⁵, Domenico Ribatti⁴⁴, Curzio Rüegg⁴⁵, Reinier O. Schlingemann¹⁸, Reinier O. Schlingemann³⁴, Stefan Schulte-Merker, Lois E.H. Smith¹³, Jonathan W. Song⁴⁶, Steven A. Stacker⁴⁷, Jimmy Stalin, Amber N. Stratman¹⁶, Maureen Van de Velde³⁶, Victor W.M. van Hinsbergh¹⁸, Peter B. Vermeulen⁴⁸, Johannes Waltenberger⁴⁹, Brant M. Weinstein¹⁶, Hong Xin²⁶, Bahar Yetkin-Arik³⁴, Seppo Ylä-Herttuala³⁷, Mervin C. Yoder³⁹, Arjan W. Griffioen⁹ - Show less +87 more•Institutions (49)

University of Geneva¹, University of Helsinki², Katholieke Universiteit Leuven³, University of Washington⁴, University of Wisconsin-Madison⁵, German Cancer Research Center⁶, Heidelberg University⁷, University of Nottingham⁸, VU University Amsterdam⁹, University of California, Irvine¹⁰, University of California, San Francisco¹¹, French Institute of Health and Medical Research¹², Harvard University¹³, Maine Medical Center¹⁴, University of Turin¹⁵, National Institutes of Health¹⁶, University of Texas Southwestern Medical Center¹⁷, University of Lausanne¹⁸, University of Missouri¹⁹, École Polytechnique Fédérale de Lausanne²⁰, Uppsala University²¹, University of Arkansas for Medical Sciences²², University of Bern²³, University of Virginia²⁴, Imperial College London²⁵, University of California, San Diego²⁶, University College London²⁷, Flanders Institute for Biotechnology²⁸, University of Oxford²⁹, University of California, Los Angeles³⁰, University of Texas MD Anderson Cancer Center³¹, University of Toronto³², University of Illinois at Chicago³³, University of Amsterdam³⁴, George Washington University³⁵, University of Liège³⁶, University of Eastern Finland³⁷, Ludwig Institute for Cancer Research³⁸, Indiana University³⁹, University of Edinburgh⁴⁰, Maastricht University⁴¹, Loyola University Medical Center⁴², National Scientific and Technical Research Council⁴³, University of Bari⁴⁴, University of Fribourg⁴⁵, Ohio State University⁴⁶, University of Melbourne⁴⁷, University of Antwerp⁴⁸, University of Münster⁴⁹

01 Aug 2018-Angiogenesis

TL;DR: In vivo, ex vivo, and in vitro bioassays that are available for the evaluation of angiogenesis are described and critical aspects that are relevant for their execution and proper interpretation are highlighted.

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Abstract: The formation of new blood vessels, or angiogenesis, is a complex process that plays important roles in growth and development, tissue and organ regeneration, as well as numerous pathological conditions. Angiogenesis undergoes multiple discrete steps that can be individually evaluated and quantified by a large number of bioassays. These independent assessments hold advantages but also have limitations. This article describes in vivo, ex vivo, and in vitro bioassays that are available for the evaluation of angiogenesis and highlights critical aspects that are relevant for their execution and proper interpretation. As such, this collaborative work is the first edition of consensus guidelines on angiogenesis bioassays to serve for current and future reference.

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397 citations

Book Chapter•DOI•

In Vitro Cytotoxicity and Cell Viability Assays: Principles, Advantages, and Disadvantages

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Özlem Sultan Aslantürk

20 Dec 2017

TL;DR: In this chapter, information is given about in vitro cytotoxicity and viability assays, these assays will be classified and their advantages and disadvantages will be emphasized.

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Abstract: 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.

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183 citations

Cites background or methods from "Cell Viability Assays"

...The incubation period required to generate a sufficient fluorescent signal above background is usually about 1–4 hours, depending on metabolic activity of the cells, the cell density per well and other conditions such as the culture medium type [54]....
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...In these cells, cytoplasmic aminopeptidase activity removes the gly and phe amino acids to release aminofluorocoumarin (AFC) and produce a fluorescent signal proportional to the number of viable cells [54]....
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...This also eliminates a plate handling step [54]....
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...When cells damaged lethally and lose membrane integrity, they lose the ability to synthetize ATP and the ATP level of cells decreases dramatically [54, 63]....
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...Furthermore, the incubation time is much shorter (30 min-1 hour) compared to 1–4 hours required for the tetrazolium assays [54]....
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Journal Article•DOI•

Biocompatibility characteristics of the metal organic framework ZIF-8 for therapeutical applications

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Marcus Hoop¹, Claudio F. Walde¹, Raffaele Ricco², Fajer Mushtaq¹, Anastasia Terzopoulou¹, Xiang-Zhong Chen¹, Andrew J. deMello¹, Christian J. Doonan³, Paolo Falcaro², Bradley J. Nelson¹, Josep Puigmartí-Luis¹, Salvador Pané¹ - Show less +8 more•Institutions (3)

ETH Zurich¹, Graz University of Technology², University of Adelaide³

01 Jun 2018-Applied Materials Today

TL;DR: A clear understanding is established of the interaction of ZIF-8 and its constituents with various cell lines and highlights the important biocompatibility factors that must be considered for future in vivo testing.

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162 citations

Journal Article•DOI•

Hallmarks of senescence and aging.

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Slavica Dodig¹, Ivana Čepelak¹, Ivan Pavić¹•Institutions (1)

University of Zagreb¹

15 Oct 2019-Biochemia Medica

TL;DR: It is considered that the telomere length is the weak biomarker (with poor predictive accuracy), and there is currently no reliable biomarker that meets all the necessary criteria.

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Abstract: The complex process of biological aging, as an intrinsic feature of living beings, is the result of genetic and, to a greater extent, environmental factors and time. For many of the changes taking place in the body during aging, three factors are important: inflammation, immune aging and senescence (cellular aging, biological aging). Senescence is an irreversible form of long-term cell-cycle arrest, caused by excessive intracellular or extracellular stress or damage. The purpose of this cell-cycles arrest is to limit the proliferation of damaged cells, to eliminate accumulated harmful factors and to disable potential malignant cell transformation. As the biological age does not have to be in accordance with the chronological age, it is important to find specific hallmarks and biomarkers that could objectively determine the rate of age of a person. These biomarkers might be a valuable measure of physiological, i.e. biological age. Biomarkers should meet several criteria. For example, they have to predict the rate of aging, monitor a basic process that underlies the aging process, be able to be tested repeatedly without harming the person. In addition, biomarkers have to be indicators of biological processes, pathogenic processes or pharmacological responses to therapeutic intervention. It is considered that the telomere length is the weak biomarker (with poor predictive accuracy), and there is currently no reliable biomarker that meets all the necessary criteria.

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146 citations

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References

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Open Access

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Journal Article•DOI•

Human malaria parasites in continuous culture

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William Trager¹, James B. Jensen¹•Institutions (1)

Rockefeller University¹

20 Aug 1976-Science

TL;DR: Plasmodium falciparum can now be maintained in continuous culture in human erythrocytes incubated at 38 degrees C in RPMI 1640 medium with human serum under an atmosphere with 7 percent carbon dioxide and low oxygen.

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Abstract: Plasmodium falciparum can now be maintained in continuous culture in human erythrocytes incubated at 38 degrees C in RPMI 1640 medium with human serum under an atmosphere with 7 percent carbon dioxide and low oxygen (1 or 5 percent). The original parasite material, derived from an infected Aotus trivirgatus monkey, was diluted more than 100 million times by the addition of human erythrocytes at 3- or 4-day intervals. The parasites continued to reproduce in their normal asexual cycle of approximately 48 hours but were no longer highly synchronous. The have remained infective to Aotus.

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7,496 citations

"Cell Viability Assays" refers background or methods in this paper

...The purpose of this chapter is to describe the detailed practical procedures behind three complementary techniques (volume, resazurin reduction, and acid phosphatase activity) for spheroid viability assessment in high-throughput 96-well format [1]....
[...]
...Cytotoxicity is expressed as a concentration-dependent reduction of the uptake of NR after exposure to the xenobiotic, thus providing a sensitive, integrated signal of both cell integrity and cell growth inhibition [1]....
[...]
...Especially during cultivation conditions like the availability of glucose [1] or a change in pH [32] influence the reliability of the MTT assay....
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...The practical application of these methods and the characterization of linearity and sensitivity have been discussed in our recent publication [1]....
[...]
...Avoid refreezing of thawed aliquots to prevent accumulation of formazan by unspecific conversion of MTT [1]....
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Journal Article•DOI•

Synchronization of Plasmodium falciparum erythrocytic stages in culture.

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Chris Lambros, Jerome P. Vanderberg

01 Jun 1979-Journal of Parasitology

TL;DR: Synchronous development of the erythrocytic stages of a human malaria parasite, Plasmodium falciparum, in culture was accomplished by suspending cultured parasites in 5% D-sorbitol and subsequent reintroduction into culture.

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Abstract: Synchronous development of the erythrocytic stages of a human malaria parasite, Plasmodium falciparum, in culture was accomplished by suspending cultured parasites in 5% D-sorbitol and subsequent reintroduction into culture. Immediately after sorbitol treatment, cultures consisted mainly of single and multiple ring-form infections. At the same time, varying degrees of lysis of erythrocytes infected with the more mature stages of the parasite was evident. Approximately 95% of the parasites were in the ring stage of development at 48 and 96 hr after sorbitol treatment-likewise, a high percentage of trophozoite and schizont stages was observed at 24, 72, and 120 hr. D-Mannitol produced similar, selective, lytic effects.

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3,387 citations

"Cell Viability Assays" refers background or methods in this paper

...We have used the three viability assays in a variety of human medulloblastoma cells lines [26], patient-derived early-passage medulloblastoma cultures, and human fetal brain-derived neurospheres [1, 19]....
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...In cocultures, the volume or metabolic activity of the coculture spheroid can be used as a proxy measure for the total number of cells; subsequently the proportion of each cell type can be quantified with microscopy or flow cytometry [19]....
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...These include cell viability [19, 20], culture proliferation, or cytotoxicity studies [21] and to a certain extent also high-throughput screenings [22, 23]....
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...The resazurin assay does not kill or lyse the spheroids and the latter can be harvested for dissociation and cell counts, flow cytometry, or histology 4h after resazurin addition [1, 19]....
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...High-throughput plate- based image cytometry systems such as the Celigo [18] (Nexcelom Bioscience), Opera [19, 20] (Perkin Elmer), and IN Cell Analyzer 2200 [20, 21] (GE) have also been developed to measure cell viability in standard multiwell microplates....
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Journal Article•DOI•

Quantitative assessment of antimalarial activity in vitro by a semiautomated microdilution technique.

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R E Desjardins, Craig J. Canfield, J D Haynes, J D Chulay

01 Dec 1979-Antimicrobial Agents and Chemotherapy

TL;DR: A rapid, semiautomated microdilution method was developed for measuring the activity of potential antimalarial drugs against cultured intraerythrocytic asexual forms of the human malaria parasite Plasmodium falciparum, and results demonstrated that the method is sensitive and precise.

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Abstract: A rapid, semiautomated microdilution method was developed for measuring the activity of potential antimalarial drugs against cultured intraerythrocytic asexual forms of the human malaria parasite Plasmodium falciparum. Microtitration plates were used to prepare serial dilutions of the compounds to be tested. Parasites, obtained from continuous stock cultures, were subcultured in these plates for 42 h. Inhibition of uptake of a radiolabeled nucleic acid precursor by the parasites served as the indicator of antimalarial activity. Results of repeated measurements of activity with chloroquine, quinine, and the investigational new drug mefloquine demonstrated that the method is sensitive and precise. Several additional antimalarial drugs and compounds of interest were tested in vitro, and the results were consistent with available in vivo data. The use of P. falciparum isolates with known susceptibility to antimalarial drugs also permitted evaluation of the cross-resistance potential of each compound tested. The applications and expectations of this new test system within a drug development program are discussed.

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2,474 citations

"Cell Viability Assays" refers background or methods in this paper

...The turnover rate can be evaluated by selective reduction of certain compounds, such as different tetrazolium salts (MTT, MTS, XTT, or WST) or resazurin as the enzymatic reduction of these compounds by dehydrogenases uses NADH/ NADPH as co-substrate....
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...Once NR is in the cell, it accumulates intracellularly in lysosomes, where a proton gradient assures a more acidic pH and the dye becomes charged [2]....
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...Spheroids are self-organized three-dimensional (3D) aggregates of cells displaying physiologically relevant gradients of oxygen, nutrients, and cell–cell and cell matrix interactions [2, 3]....
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...The amount of reduced WST-tetrazolium can be quantified with an absorption measurement at 450 nm in the culture medium....
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...These game-changing inventions established the application of flow cytometry into virtually any biomedical field as well as in clinical settings [2]....
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Journal Article•DOI•

Neutral red uptake assay for the estimation of cell viability/cytotoxicity.

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Guillermo Repetto, Ana del Peso, Jorge L. Zurita

01 Jun 2008-Nature Protocols

TL;DR: The neutral red uptake assay provides a quantitative estimation of the number of viable cells in a culture and is cheaper and more sensitive than other cytotoxicity tests (tetrazolium salts, enzyme leakage or protein content).

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Abstract: The neutral red uptake assay provides a quantitative estimation of the number of viable cells in a culture. It is one of the most used cytotoxicity tests with many biomedical and environmental applications. It is based on the ability of viable cells to incorporate and bind the supravital dye neutral red in the lysosomes. Most primary cells and cell lines from diverse origin may be successfully used. Cells are seeded in 96-well tissue culture plates and are treated for the appropriate period. The plates are then incubated for 2 h with a medium containing neutral red. The cells are subsequently washed, the dye is extracted in each well and the absorbance is read using a spectrophotometer. The procedure is cheaper and more sensitive than other cytotoxicity tests (tetrazolium salts, enzyme leakage or protein content). Once the cells have been treated, the assay can be completed in <3 h.

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1,701 citations

Journal Article•DOI•

Simple and Inexpensive Fluorescence-Based Technique for High-Throughput Antimalarial Drug Screening

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Martin J. Smilkstein¹, Nongluk Sriwilaijaroen², Jane Xu Kelly³, Prapon Wilairat², Michael K. Riscoe³ - Show less +1 more•Institutions (3)

Oregon Health & Science University¹, Mahidol University², United States Department of Veterans Affairs³

01 May 2004-Antimicrobial Agents and Chemotherapy

TL;DR: A side-by-side comparison of this new fluorescence assay and a standard radioisotopic method suggest that it may be an ideal method for high-throughput antimalarial drug screening.

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Abstract: Radioisotopic assays involve expense, multistep protocols, equipment, and radioactivity safety requirements which are problematic in high-throughput drug testing. This study reports an alternative, simple, robust, inexpensive, one-step fluorescence assay for use in antimalarial drug screening. Parasite growth is determined by using SYBR Green I, a dye with marked fluorescence enhancement upon contact with Plasmodium DNA. A side-by-side comparison of this fluorescence assay and a standard radioisotopic method was performed by testing known antimalarial agents against Plasmodium falciparum strain D6. Both assay methods were used to determine the effective concentration of drug that resulted in a 50% reduction in the observed counts (EC(50)) after 48 h of parasite growth in the presence of each drug. The EC(50)s of chloroquine, quinine, mefloquine, artemisinin, and 3,6-bis-epsilon-(N,N-diethylamino)-amyloxyxanthone were similar or identical by both techniques. The results obtained with this new fluorescence assay suggest that it may be an ideal method for high-throughput antimalarial drug screening.

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1,008 citations

"Cell Viability Assays" refers background or methods in this paper

...To overcome this time-consuming post-reaction processing some tetrazolium derivatives that produce water-soluble products have been developed, such as MTS (3-(4,5-dimethylthiazol-2-yl)5-(3- carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) [9, 10], XTT (2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2Htetrazolium5-carboxanilide) [11, 12], or WST (2-(2-methoxy-4nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2Htetrazolium) [13, 14]....
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...[8, 9] has enabled researchers to produce a single spheroid per well and control spheroid size in a high-throughput format....
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...Nevertheless, only a minority of chemicals have yet been tested for DNT [8, 9],...
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