Resazurin is blue and nonfluorescent whereas resorufin is red and highly fluorescent. Trek diagnostic systems, in comparison with these other methods of susceptibility testing, may have many potential advantages. Evaluation of rapid microplate assays using cellular. No other manual has been so popular, or so influential. This protocol takes you stepbystep through the use of alamarblue reagent to monitor viability in mammalian cells using a. In vitro toxicology assay kit resazurin based stock no. A new rapid and simple nonradioactive assay to monitor and determine the proliferation of lymphocytes. The optimum cell density may vary between cell types. This results in colorimetric absorbance and fluorescence changes. It is a nontoxic, water soluble, redoxsensitive dye that changes from its bluenonfluorescent state to a pinkhighlyfluorescent state upon reduction by viable cells. I have a problem understanding connecting absorbance with. A novel onestep, highly sensitive fluorometric assay to evaluate cellmediated cytotoxicity. Being a nonproprietary product and cheaper than alamar blue, it could be easily implemented in lowresource settings.
Optimized alamarblue assay protocol for drug doseresponse. Alamarblue assay definition of alamarblue assay by. On the other hand, the alamarblue assay all rights belong to thermofisher is soluble in media, is nontoxic and can be applied to cells multiple times throughout the. An assay used to quantify the proliferation of various human and animal cell lines, bacteria and fungi, and assess relative cytotoxicity of agents within various chemical classes. It is a proven safe and nontoxic dye used for quantitative analysis of cell viability and cell proliferation, for. Adjust the cell count to 1 x 10 4 cellsml suggested cell density. Compared to alamarblue, alamarblue hs contains highly purified resazurin and provides higher sensitivity, and a larger assay window.
Tox8 store at 28ec this kit is designed for fluorometrically or spectrophotometrically determining cell number as a function of metabolic activity using the dye resazurin. Resazurin microtiter assay plate method for detection of. Alamar blue protocol nov302006 has any one done the cell viability cell toxicity assay using alamar blue. L collins and s g franzblau pharmacology research department, gillis w. Depending on the cell type, the resazurin assay can be used to detect as few as 40 cells with reproducible and sensitive signal. The results were compared to those of the proportion method pm on lj. For example, ab results can be read visually, spectrofluorometrically or spectrophotometrically, its reduction is dependent on active metabolism, it is amenable to highthroughput, its use is cost effective and ab lacks toxicity to humans and microorganisms.
By monitoring the absorbance at 570 nm and 600 nm, relative metabolic activity for the cells can be determined. Nanoparticles nps are known to interfere with many highthroughput cell viability and cell proliferation assays, which complicates the assessment of their potential toxic effects. Alamarblue cell viability reagent from thermo fisher. Fluorescence can be read using 544 nm excitation and 590 nm emission wavelengths, or absorbance can be read using a. Resazurin cell viability assay offers a simple, rapid, reliable, sensitive, safe and costeffective measurement of cell viability. Since alamar blue has been recently identified as resazurin in cell cytotoxicity studies, we have standardized and evaluated a microplate method which uses the reduction of resazurin for dst to inh and rif in clinical isolates of m. The absorbance spectrum for reduced and oxidized forms of the resazurin dye are highlighted in figure 1. Similarly, in the nccls planktonic assay 17, minimum bactericidal concentrations mbcs are obtained from the same wells as mics. Franzblaumicroplate alamar blue assay versus bactec 460 system for highthroughput screening of compounds against mycobacterium tuberculosis and mycobacterium avium antimicrob. Assessment of cell proliferation with resazurinbased. A simple method to measure cell viability in proliferation.
Multiple applications of alamar blue as an indicator of metabolic. The mtt assay is by far the most popular, but being insoluble in culture media, it is an endpoint assay. The 96well microplate alamar blue assay maba allows for the quantitative determination of drug susceptibility against any strain of replicating mycobacterium tuberculosis to be completed within a week at minimal cost. Resazurin dye has been broadly used as indicator of cell vi ability in several types of assays for evaluation of the biocompatibility. The bioassay may also be used to establish relative cytotoxicity of agents within various chemical classes 3. Rapid, lowtechnology cmi determination with clinical mycobacterium tuberculosis isolates by using the microplate alamar blue assay. Our customizable cdc assays accurately detect cell lysis based on alamar blue staining of remaining live cells, using human serum complement as a probe. Assessment of the alamar blue assay for cellular growth and viability in vitro. Molecular cloning, fourth edition, by the celebrated founding author joe sambrook and new coauthor, the distinguished hhmi investigator michael green, preserves the highly praised. This is a trusted and established reagent which has been available since 1993. Add cells in appropriate medium to microplate wells. Moreover, coban 2012 reported 100% agreement between the resazurin microplate assay for the rapid determination of mrsa and the cefoxitin mic.
Microplate alamar blue assay versus bactec 460 system for. The alamarblue reagent is an oxidized form of redox indicator that is blue in color and nonfluorescent. Here, we optimized the standard alamarblue proliferationviability protocol for tumor spheroid cultures to enhance assay precision during. Compounds such as tetrazolium salts and alamarblue, which can be reduced by cellular metabolic intermediates, can be used to monitor cellular proliferation. A cell proliferation alamarblue assay by qgel on vimeo, the home for high quality videos and the people who love them. Plate cells and expose to test agent as determined by. The alamar blue assay is based on enzymatic reduction of indicator dye by viable cells and serves as an effective tool for assessing cell proliferation and as a screening technique. Assessment of a simple, nontoxic alamar blue cell survival assay to monitor tomato cell viability. The assay is based on detection of metabolic activity through an oxidationreduction redox indicator, which both fluoresces and changes colour in. Alamar blue monitors the reducing environment of the living cell. Resazurin can also be used to measure the aerobic biodegradation of organic matter found in effluents. Measuring cytotoxicity or proliferation alamarblue assay. Franzblau sg, witzig rs, mcclaughlin jc, torres p, madico g, hernandez a, et al. Microplate alamar blue assay how is microplate alamar.
The alamarblue hs and alamarblue cell viability reagents are readytouse resazurinbased reagents that function as cell health indicators by using the reducing power of living cells to quantitatively measure viability. For an alamar blue assay cell viablity experiment, how do i explain or interpret the results of a absorbance vs. A 96well plate containing the cells and the compounds to be tested is prepared using standard methods. Supplementary information validation of alamarblue.
It uses the indicator dye resazurin to measure the. Microplate based alamar blue assay maba is a practical, sensitive and inexpensive assay method of cell viability in which fluorescence reduction assay of alamar blue, a resazurin a dark blue dye and nonfluoroscent in oxidized form, available as a sterile and liquid reagent commercially, is used in a microplate format. Microplate alamar blue assay versus bactec 460 system for highthroughput screening of compounds against mycobacterium tuberculosis and mycobacterium avium. Upon entering living cells, resazurin is reduced to resorufin, a compound that is red in color and highly fluorescent.
Method for measuring cytotoxicity or proliferation using alamarblue by spectrophotometry. When incubated with viable cells, the reagent changes. Alamar blue assay may242009 hi guys, im going to be doing some compound screening using the alamar blue proliferation assay in a 96 well plate format on either hek or mcf7 cells. The alamarblue assay is designed to measure quantitatively the proliferation of various human. We selected the alamar blue assay as our primary assay for reasons well articulated by earlier contributors to this discussion.
Harvest cells which are in the log phase of growth and determine cell count. Quantitate in vitro antiproliferation experiments using a. The thermo scientific alamarblue cell viability assay reagent is used to quantitatively measure the viability of mammalian cell lines, bacteria and fungi by incorporating a rapid, sensitive and reliable fluorometriccolorimetric growth indicator. One main concern with this type of test is biosafety. It can be applied in studies concentrating on animal, plant, yeast, and bacteria cells. How do you properly use alamar blue for a continuous. Resazurin can be used to assay lglutamate, achieving a sensitivity of 2. The unique properties of these materials increase the likelihood that they will interfere with analytical techniques, which may contribute to this phenomenon. Microplate alamar blue assay for susceptibility testing of.
No qc protocol is recommended for fluorescence since. The evaluation of engineered nanomaterial safety has been hindered by conflicting reports demonstrating differential degrees of toxicity with the same nanoparticles. Resazurin 7hydroxy3hphenoxazin3one 10oxide is a phenoxazine dye that is weakly fluorescent, nontoxic, cellpermeable, and redox. In vitro toxicology assay kit, resazurin based tox8. Throughput in tuberculosis drug discovery was extremely limited prior to the introduction of microplatebased susceptibility assays. The internal environment of proliferating cells is more reduced than that of nonproliferating cells.
Send us your target cell line, or use one of ours, and well take care of the rest. The alamarblue dye is a redox indicator that yields a colorimetric change and a fluorescent signal in response to metabolic activity. A laboratory manual fourth editionmolecular cloning has served as the foundation of technical expertise in labs worldwide for 30 years. A simple method to measure cell viability in proliferation scielo. Alamar blue is stable and nontoxic, so unlike other metabolic assays, it is possible to plate directly out of the wells over long periods for determination of viable cell counts. Alamarblue assay for cell proliferation bmg labtech.
The simple protocol involves adding a single reagent directly to cells cultured in serumsupplemented medium. So one ends up going round and round without any clear conclusion. Antimalarial, antimycobacterial and cytotoxic limonoids from chisocheton siamensis tuberculosis was assessed by the microplate alamar blue assay as previously described collins and franzblau, 1997. The protocol with the reagents is not very clear and cites references. The reduction of alamarblue results in a measurable color shift. It can be used multiple times on the same culture or piece of tissue.
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