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Cold Spring Harbor Protocols Jun 2018Among viability assays that depend on the conversion of substrate to chromogenic product by live cells, the MTT assay is still among one of the most versatile and...
Among viability assays that depend on the conversion of substrate to chromogenic product by live cells, the MTT assay is still among one of the most versatile and popular assays. The MTT assay involves the conversion of the water-soluble yellow dye MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] to an insoluble purple formazan by the action of mitochondrial reductase. Formazan is then solubilized and the concentration determined by optical density at 570 nm. The result is a sensitive assay with excellent linearity up to ∼10 cells per well. As with the alamarBlue assay, small changes in metabolic activity can generate large changes in MTT, allowing one to detect cell stress upon exposure to a toxic agent in the absence of direct cell death. The assay has been standardized for adherent or nonadherent cells grown in multiple wells. The protocol uses a standard 96-well plate. This can be scaled up, however, to suit a different plate format. Plate 500-10,000 cells per well in a 96-well plate. The assay has good linearity up to 10 cells.
Topics: Biological Assay; Cell Survival; Dimethyl Sulfoxide; Formazans; Sodium Dodecyl Sulfate; Solubility; Staining and Labeling; Tetrazolium Salts
PubMed: 29858338
DOI: 10.1101/pdb.prot095505 -
Methods in Molecular Biology (Clifton,... 2019A large amount of energy used for nutrient processing and cellular functions is essential for tumorigenesis. Total intracellular adenosine triphosphate (ATP) is mainly...
A large amount of energy used for nutrient processing and cellular functions is essential for tumorigenesis. Total intracellular adenosine triphosphate (ATP) is mainly generated by glycolysis and mitochondrial oxidative phosphorylation. Here, we provide a protocol for measurements of energy metabolism in cancer cells by using Seahorse XF24 Extracellular Flux analyzer. Specifically, this machine measures glycolysis by analyzing the extracellular acidification rate (ECAR) and measures mitochondrial oxidative phosphorylation on the basis of the oxygen consumption rate (OCR), through real-time and live cell analysis. This protocol is provided for researchers who are unfamiliar with the method and to aid them in carrying out the technique successfully.
Topics: Animals; Biological Assay; Cell Line; Data Analysis; Energy Metabolism; Extracellular Space; Glycolysis; Humans; Hydrogen-Ion Concentration; Mitochondria; Neoplasms; Oxidative Phosphorylation; Oxygen Consumption; Software
PubMed: 30725464
DOI: 10.1007/978-1-4939-9027-6_18 -
Molecular Biology Reports Oct 2018Apoptosis has been recognized as a type of programmed cell death connected with characteristic morphological and biochemical changes in cells. This programmed cell death... (Review)
Review
Apoptosis has been recognized as a type of programmed cell death connected with characteristic morphological and biochemical changes in cells. This programmed cell death plays an important role in the genesis of a number of physiological and pathological processes. Thus, it can be very important to detect the signs of apoptosis in a study of cellular metabolism. The present paper provides an overview of methods often being used for detecting DNA fragmentation as one of the most specific findings in apoptosis. To date, three routine assays have been developed for detecting DNA fragmentation: DNA ladder assay, TUNEL assay, and comet assay. All these methods differ in their principles for detecting DNA fragmentation. DNA ladder assay detects the characteristic "DNA ladder" pattern formed during internucleosomal cleavage of DNA. Terminal deoxynUcleotidyl transferase Nick-End Labeling (TUNEL) assay detects DNA strand breaks using terminal deoxynucleotidyl transferase catalyzing attachment of modified deoxynucleotides on the DNA strand breaks. Comet assay can be used for detecting nucleus breakdown producing single/double-strand DNA breaks. The aim of this review is to describe the present knowledge on these three methods, including optimized approaches, techniques, and limitations.
Topics: Animals; Apoptosis; Biological Assay; Comet Assay; DNA; DNA Fragmentation; Humans; In Situ Nick-End Labeling
PubMed: 30022463
DOI: 10.1007/s11033-018-4258-9 -
Current Protocols in Protein Science Feb 2015Purification of recombinant proteins for biochemical assays and structural studies is time-consuming and presents inherent difficulties that depend on the optimization...
Purification of recombinant proteins for biochemical assays and structural studies is time-consuming and presents inherent difficulties that depend on the optimization of protein stability. The use of dyes to monitor thermal denaturation of proteins with sensitive fluorescence detection enables rapid and inexpensive determination of protein stability using real-time PCR instruments. By screening a wide range of solution conditions and additives in a 96-well format, the thermal shift assay easily identifies conditions that significantly enhance the stability of recombinant proteins. The same approach can be used as an initial low-cost screen to discover new protein-ligand interactions by capitalizing on increases in protein stability that typically occur upon ligand binding. This unit presents a methodological workflow for small-scale, high-throughput thermal denaturation of recombinant proteins in the presence of SYPRO Orange dye.
Topics: Biological Assay; Fluorometry; Ligands; Protein Stability; Proteins; Temperature
PubMed: 25640896
DOI: 10.1002/0471140864.ps2809s79 -
Gene Dec 2015The MTT assay (to a less degree MTS, XTT or WST) is a widely exploited approach for measuring cell viability/drug cytotoxicity. MTT reduction occurs throughout a cell...
The MTT assay (to a less degree MTS, XTT or WST) is a widely exploited approach for measuring cell viability/drug cytotoxicity. MTT reduction occurs throughout a cell and can be significantly affected by a number of factors, including metabolic and energy perturbations, changes in the activity of oxidoreductases, endo-/exocytosis and intracellular trafficking. Over/underestimation of cell viability by the MTT assay may be due to both adaptive metabolic and mitochondrial reprogramming of cells subjected to drug treatment-mediated stress and inhibitor off-target effects. Previously, imatinib, rottlerin, ursolic acid, verapamil, resveratrol, genistein nanoparticles and some polypeptides were shown to interfere with MTT reduction rate resulting in inconsistent results between the MTT assay and alternative assays. Here, to test the under/overestimation of viability by the MTT assay, we compared results derived from the MTT assay with the trypan blue exclusion assay after treatment of glioblastoma U251, T98G and C6 cells with three widely used inhibitors with the known direct and side effects on energy and metabolic homeostasis - temozolomide (TMZ), a DNA-methylating agent, temsirolimus (TEM), an inhibitor of mTOR kinase, and U0126, an inhibitor of MEK1/2 kinases. Inhibitors were applied shortly as in IC50 evaluating studies or long as in studies focusing on drug resistance acquisition. We showed that over/underestimation of cell viability by the MTT assay and its significance depends on a cell line, a time point of viability measurement and other experimental parameters. Furthermore, we provided a comprehensive survey of factors that should be accounted in the MTT assay. To avoid result misinterpretation, supplementation of the tetrazolium salt-based assays with other non-metabolic assays is recommended.
Topics: Animals; Biological Assay; Cell Count; Cell Line, Tumor; Cell Survival; Dacarbazine; Female; Formazans; Humans; Inhibitory Concentration 50; Rats; Rats, Wistar; Sirolimus; Temozolomide; Tetrazolium Salts
PubMed: 26260013
DOI: 10.1016/j.gene.2015.08.009 -
SLAS Discovery : Advancing Life... Jun 2019Many factors must be considered during the optimization of an enzyme assay. These include the choice of buffer and its composition, the type of enzyme and its...
Many factors must be considered during the optimization of an enzyme assay. These include the choice of buffer and its composition, the type of enzyme and its concentration, as well as the type of substrate and concentrations, the reaction conditions, and the appropriate assay technology. The process of an enzyme assay optimization, in our experience, can take more than 12 weeks using the traditional one-factor-at-a-time approach. In contrast, the design of experiments (DoE) approaches have the potential to speed up the assay optimization process and provide a more detailed evaluation of tested variables. However, not all researchers are aware of DoE approaches or believe that it is easy to employ a DoE approach for the optimization of an assay. In order to facilitate enzyme assay developers to use DoE methodologies, we present in detail the steps required to identify in less than 3 days (1) the factors that significantly affect the activity of an enzyme and (2) the optimal assay conditions using a fractional factorial approach and response surface methodology. This is exemplified with the optimization of assay conditions for the human rhinovirus-3C protease, and the methodology used could be employed as a basic guide for the speedy identification of the optimum assay conditions for any enzyme.
Topics: 3C Viral Proteases; Biological Assay; Cysteine Endopeptidases; Enzyme Assays; Humans; Research Design; Substrate Specificity; Viral Proteins
PubMed: 30802413
DOI: 10.1177/2472555219830084 -
Cold Spring Harbor Protocols Jun 2018This protocol describes viability measurements for cell cultures in a 96-well tissue culture plate using alamarBlue (resazurin). The assay can be modified to accommodate...
This protocol describes viability measurements for cell cultures in a 96-well tissue culture plate using alamarBlue (resazurin). The assay can be modified to accommodate larger plates; however, for a preliminary analysis of transfection reagents and parameters of the transfection protocol on cell viability, a 96-well plate format is the most cost effective.
Topics: Biological Assay; Cell Survival; Cells, Cultured; Oxazines; Xanthenes
PubMed: 29858336
DOI: 10.1101/pdb.prot095489 -
Basic & Clinical Pharmacology &... May 2021Predictive biomarkers play an important role in our efforts to individualize pharmacotherapy, and within recent years, a number of different types of assays have been... (Review)
Review
Predictive biomarkers play an important role in our efforts to individualize pharmacotherapy, and within recent years, a number of different types of assays have been introduced. These biomarkers may potentially support the selection and dosage of specific drugs in order to maximize efficacy and minimize adverse reactions in the individual patient. However, in many instances, the scientific and clinical evidence is insufficient to support the prescribing decision. When predictive biomarkers are used to guide pharmacotherapy, it is important to secure that decisions are based on solid clinical evidence. Here, the regulatory authorities, especially the FDA, have been at the forefront in relation to regulate this type of biomarker assay in order to secure patient safety. The approval process for companion diagnostics is an example of this effort, where the scientific validity of the biomarker and assay is in focus. With the approaching implementation of the new IVD Regulation, greater attention will also be paid to analytical and clinical validity of biomarker assays in the EU. For any type of predictive biomarker assay, including pharmacogenetic and tumour profiling tests, the clinical evidence needs to be in place before they are used routinely in the clinic.
Topics: Biological Assay; Biomarkers; Diagnostic Test Approval; European Union; Pharmacogenomic Testing; Precision Medicine; Reagent Kits, Diagnostic; United States; United States Food and Drug Administration
PubMed: 33665955
DOI: 10.1111/bcpt.13578 -
Bioanalysis Jul 2023The 2022 16th Workshop on Recent Issues in Bioanalysis (WRIB) took place in Atlanta, GA, USA on September 26-30, 2022. Over 1000 professionals representing...
2022 White Paper on Recent Issues in Bioanalysis: FDA Draft Guidance on Immunogenicity Information in Prescription Drug Labeling, LNP & Viral Vectors Therapeutics/Vaccines Immunogenicity, Prolongation Effect, ADA Affinity, Risk-based Approaches, NGS, qPCR, ddPCR Assays ( - Recommendations on Gene...
The 2022 16th Workshop on Recent Issues in Bioanalysis (WRIB) took place in Atlanta, GA, USA on September 26-30, 2022. Over 1000 professionals representing pharma/biotech companies, CROs, and multiple regulatory agencies convened to actively discuss the most current topics of interest in bioanalysis. The 16th WRIB included 3 Main Workshops and 7 Specialized Workshops that together spanned 1 week in order to allow exhaustive and thorough coverage of all major issues in bioanalysis, biomarkers, immunogenicity, gene therapy, cell therapy and vaccines. Moreover, in-depth workshops on ICH M10 BMV final guideline (focused on this guideline training, interpretation, adoption and transition); mass spectrometry innovation (focused on novel technologies, novel modalities, and novel challenges); and flow cytometry bioanalysis (rising of the 3rd most common/important technology in bioanalytical labs) were the special features of the 16th edition. As in previous years, WRIB continued to gather a wide diversity of international, industry opinion leaders and regulatory authority experts working on both small and large molecules as well as gene, cell therapies and vaccines to facilitate sharing and discussions focused on improving quality, increasing regulatory compliance, and achieving scientific excellence on bioanalytical issues. This 2022 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2022 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 3) covers the recommendations on Gene Therapy, Cell therapy, Vaccines and Biotherapeutics Immunogenicity. Part 1 (Mass Spectrometry and ICH M10) and Part 2 (LBA, Biomarkers/CDx and Cytometry) are published in volume 15 of Bioanalysis, issues 16 and 15 (2023), respectively.
Topics: Prescription Drugs; Technology; Biological Assay; Biomarkers; Cell- and Tissue-Based Therapy
PubMed: 37526071
DOI: 10.4155/bio-2023-0135 -
Annual Review of Analytical Chemistry... Jul 2021Adverse effects of environmental toxicants to human health have traditionally been assayed using in vitro assays. Organ-on-chip (OOC) is a new platform that can bridge... (Review)
Review
Adverse effects of environmental toxicants to human health have traditionally been assayed using in vitro assays. Organ-on-chip (OOC) is a new platform that can bridge the gaps between in vitro assays (or 3D cell culture) and animal tests. Microenvironments, physical and biochemical stimuli, and adequate sensing and biosensing systems can be integrated into OOC devices to better recapitulate the in vivo tissue and organ behavior and metabolism. While OOCs have extensively been studied for drug toxicity screening, their implementation in environmental toxicology assays is minimal and has limitations. In this review, recent attempts of environmental toxicology assays using OOCs, including multiple-organs-on-chip, are summarized and compared with OOC-based drug toxicity screening. Requirements for further improvements are identified and potential solutions are suggested.
Topics: Animals; Biological Assay; Ecotoxicology; Humans; Lab-On-A-Chip Devices; Oligonucleotide Array Sequence Analysis
PubMed: 33974806
DOI: 10.1146/annurev-anchem-091620-091335