A simple method to measure cell viability in proliferation and cytotoxicity assays abstract. Optimized alamarblue assay protocol for drug doseresponse. 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. Method for measuring cytotoxicity or proliferation using alamarblue by spectrophotometry. Cytotoxicity against human sf268 cells after 72 hrs by alamar. The continued growth of viable cells maintain a reducing environment fluorescent, red and inhibition of growth maintains an oxidized environment nonfluorescent, blue, which can be detected using a. The following features and benefits establish alamarblue as a preferred alternative reagent to similar cell proliferation assays mtt. The bioassay may also be used to establish relative cytotoxicity of agents within various chemical classes 3.
It can also be used to study mycobacteria, bacteria and fungi. Determination of ocular irritation potential in vivo assay of resazurin and of benzalkonium chloride seven albino rabbits 2. Alamarblue assay for cell proliferation bmg labtech. Franzblaumicroplate alamar blue assay versus bactec 460 system for highthroughput screening of compounds against mycobacterium tuberculosis and mycobacterium avium antimicrob. Plate cells in 96well plate black plate with clear bottom.
Resazurin dye has been broadly used as indicator of cell viability in several types of assays for evaluation of the biocompatibility of medical and dental materials. Multiple applications of alamar blue as an indicator of. Resazurin 7hydroxy3hphenoxazin3one 10oxide is a phenoxazine dye that is weakly fluorescent, nontoxic, cellpermeable, and redox. Here, we optimized the standard alamarblue proliferationviability protocol for tumor spheroid cultures to enhance assay precision during. The following features and benefits establish alamarblue as a preferred alternative reagent to similar cell proliferation assays. Plate cells and expose to test agent as determined by. Alamar blue protocol nov302006 has any one done the cell viability cell toxicity assay using alamar blue. Resazurin reduction assay alamar blue resazurin 7hydroxy3hphenoxazin3one10oxide is a blue dye, which is reduced to pink fluorescent resorufin in the presence of mitochondrial enzymes. Resazurin cell viability kit protocol specific for.
Resazurin metabolism assay is a new sensitive alternative. How to perform a dualfluorescent aoeb assay video demonstration duration. The cell viability assay by alamar blue is based on the principle of reduction of the nonfluorescent reagent resazurin to a fluorescent compound resarufin by the intracellular. Assays that measure metabolic activity are suitable for analyzing proliferation, viability, and cytotoxicity. Onetenth of culture volume of the celltiterblue solution was added to each well of the 96well.
After viability determination, the diluted alamarblue hs or alamarblue reagent can be replaced with complete media and returned to the incubator. The reduction of tetrazolium salts such as mtt, xtt, and wst1 to colored formazan compounds or the bioreduction of resazurin occurs only in metabolically active cells. It is a proven safe and nontoxic dye used for quantitative analysis of cell viability and cell proliferation, for cytokine bioassays and in vitro cytotoxicity studies. Alamar blue commercial solution were wavelength scanned using the spectrophotometer.
The goal was to make the biofilm susceptibility assay as similar to the nccls planktonic susceptibility assay 17 as possible. Alamarblue assay definition of alamarblue assay by. 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. 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. The active ingredient of alamarblue resazurin is a nontoxic figure 1, cell permeable compound that is blue in color and virtually nonfluorescent. Results are expressed as mean percentage of viability compared to nontreated controls 100%, dotted line.
Microplate alamar blue assay maba and low oxygen recovery. Microplate alamar blue assay for staphylococcus epidermidis. 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. The optimum cell density may vary between cell types. Singlestep, homogeneous, highthroughput cell quantitation.
The main object of our study was to investigate whether the resazurin metabolism assay is a sensitive surfactant and alcohol toxicity test in isolated pig cornea and to compare this recently developed fluorometric assay with the data collected in the eye irritation reference chemical data bank. A simple method to measure cell viability in proliferation. Plate cells and expose to test agent as determined by researcher. Prestoblue cell viability reagent can be used with cells plated in their standard growth media containing serum and phenol red. Choosing the correct method for conducting cell viability measurement is essential for obtaining consistent accurate results. When reduced by metabolically active cells the nonfluorescent dark blue dye becomes fluorescent pink with absorbance at 570nm and redfluorescent properties 560ex590em at neutral ph. Tmre is the most popular abcam mitochondrial membrane dye assay.
Nonviable cells rapidly lose metabolic capacity, do not reduce the indicator dye, and thus do not generate a fluorescent signal. L, relatively nontoxic and provided that it is carried out carefully, the same replicates. General method for measuring cytotoxicity or proliferation using alamarblue harvest cells which are in the log phase of growth and determine cell count. Cytotoxicity against human jurkattall cells after 48 hrs by alamar blue assay. Viable cells retain the ability to reduce resazurin into resorufin.
So one ends up going round and round without any clear conclusion. Adjust the cell count to 1 x 10 4 cellsml suggested cell density. You will have to determine the length of time for development though. Measuring cytotoxicity or proliferation alamarblue assay. Compared to alamarblue, alamarblue hs contains highly purified resazurin and provides higher sensitivity, and a larger assay window. Viable cells continuously convert resazurin to resorufin, thereby generating a quantitative measure of viabilityand cytotoxicity.
Just dilute into your cell growth media and place on your cells. It is a nontoxic, water soluble, redoxsensitive dye that changes from its blue nonfluorescent state to a pinkhighlyfluorescent state upon reduction by viable cells. Upon entering living cells, resazurin is reduced to resorufin, a compound that is red in color and highly fluorescent. Prestoblue reagent performance compared to alamarblu and celltiterblue figure 4. Methods for highthroughput screening of antibiofilm compounds are needed. Resazurin microtiter assay plate method for detection of. Harvest cells which are in the log phase of growth and determine cell count.
Description the celltiter blue cell viability assay provides a homogeneous. L, relatively nontoxic and provided that it is carried out carefully, the same replicates can be followed over several time points. Trypan blue dye exclusion assay is based on the principle that live cells possess intact cell membranes that exclude this dye, whereas dead cells. No qc protocol is recommended for fluorescence since. Aid 1056617 cytotoxicity against human sf268 cells after. Check to make sure your cell densities are in the linear range of the assay. The assay is based on detection of metabolic activity through an oxidationreduction redox indicator, which both fluoresces and changes colour in. Cell viability and proliferation assays sigmaaldrich. It uses the indicator dye resazurin to measure the metabolic capacity of cellsan indicator of cell viability. Three rabbits were used for the determination of resazurin in vivo ocular irritation index. In alamarblue assay the growing cells cause a chemical reduction of the alamarblue dye from nonfluorescent blue to red fluorescent.
Cytotoxicity against human sf268 cells after 72 hrs by alamar blue assay. The alamarblue dye is a redox indicator that yields a colorimetric change and a fluorescent signal in response to metabolic activity. This dye exclusion assay is used to determine the number of viable andor dead cells in a cell suspension. Alamarblue cell viability reagent from thermo fisher. Throughput in tuberculosis drug discovery was extremely limited prior to the introduction of microplatebased susceptibility assays. Viability using trypan blue introduction since the quality of the cell sample is vital for potential downstream experiments, viability measurements are routinely performed in many laboratories. This is a trusted and established reagent which has been available since 1993. The alamarblue reagent allows for viability studies of chemical inhibitors on tumor cell lines and toxicology research to establish baseline data for predicting the toxicity of. The celltiterblue assay is based on the ability of living cells to convert a redox dye resazurin into a fluorescent end product resorufin. Living cells are metabolically active and are able to reduce via mitochondrial reductase, the nonfluorescent dye resazurin to the stronglyfluorescent dye resorufin fig. Aid 753934 cytotoxicity against human jurkattall cells. A variety of parameters for the ab assay were standardized at the outset using s. It is a nontoxic, water soluble, redoxsensitive dye that changes from its bluenonfluorescent state to a pinkhighlyfluorescent state upon reduction by viable cells. The simple protocol involves adding a single reagent directly to cells cultured in serumsupplemented medium.
Multiple applications of alamar blue as an indicator of metabolic. Actively proliferating cells increase their metabolic activity, while. Due to the fact that it is extremely stable and more importantly nontoxic to the cells, continuous monitoring of cultures over time is possible ahmed et al. General method for measuring cytotoxicity or proliferation using alamarblue. Alamar blue ab is a watersoluble dye that has been previously used for quantifying in vitro viability of various cells fields and lancaster, 1993. A 96well plate containing the cells and the compounds to be tested is prepared using standard methods. Stem cells international hindawi publishing corporation.
This reduction can be quantified via fluorescence spectroscopy with peak absorption of resazurin at 600 nm and resorufin at 570 nm. Jc1 exem 530530570 and jc10 exem 590520570 form red. Alamarblue assay definition of alamarblue assay by medical. It can be applied in studies concentrating on animal, plant, yeast, and bacteria cells. The alamarblue assay is designed to measure quantitatively the proliferation of various human. Add cells in appropriate medium to microplate wells. Fluorescence can be read using 544 nm excitation and 590 nm emission wavelengths, or absorbance can be read using a spectrophotometer set at 570 nm. For absorbance reads, it is recommended that cells be incubated at least 20 minutes with the prestoblue reagent. The absorbance spectrum for reduced and oxidized forms of the resazurin dye are highlighted in figure 1. Assessment of cell proliferation with resazurinbased. Perform an initial experiment to check the best linear range for your cell type and experimental set up. A new nondestructive cytometric assay based on resazurin. The alamar blue assay in conjunction with amended agar assays would be able to discriminate among test compounds that affect spore viability, those that only suppress mycelial growth or both.
Sep 10, 2012 these findings suggest that the alamar blue assay may not be appropriate for studying proliferation in tendonderived cells. The assay is homogenous, requires no cell lysis or washing and offers a simple, rapid, reliable, sensitive, safe and costeffective measurement of. These assays are used to test cytotoxicity and cytostatic activity. By monitoring the absorbance at 570 nm and 600 nm, relative metabolic activity for the cells can be determined. Too few cells may fall below the limit of detection, and too many cells may saturate the od. Resazurin cell viability assay offers a simple, rapid, reliable, sensitive, safe and costeffective measurement of cell viability. The resazurin assay protocol uses an indicator dye to measure oxidationreduction reactions which principally occur in the mitochondria of live cells. Cell proliferation was assessed by comparing the results of an alamar blue assay and two dna quantification assays, cyquant and picogreen, on two human cancer cell lines and two human primary cell sources. The resazurin assay also known as alamar blue assay offers a simple, rapid, and sensitive measurement for the viability of mammalian cells and bacteria. Dont let the color get really pink or you will have saturated the assay.
The protocol with the reagents is not very clear and cites references. Thaw out resazurin solution if kept frozen and warm it to 37c to ensure all components are completely in solution. Resazurin is blue and nonfluorescent whereas resorufin is red and highly fluorescent. Upon entering cells, resazurin is reduced to resorufin, which produces very bright red fluorescence figure 2. It is a sensitive assay if working with higher than 5x10 3 cells per 100. This assay has excellent performance compared to other resazurinbased cell proliferation kits such as alamarblue, prestoblue, or celltiterblue. It is a fluorometric method to estimate the number of viable cells by measuring the reduction of resazurin into resorufin. Trypan blue is a large negatively charged molecule. The assay is homogenous, requires no cell lysis or washing and offers a simple, rapid, reliable, sensitive, safe and costeffective measurement of cell viability. Alamar blue monitors the reducing environment of the living cell. Readout was after 5 or 6 days with the wst1 assay wst, celltiterglo assay ctg, celltiterfluor assay ctf, alamar blue assay with absorbance aba, alamar blue assay with fluorescence abf, crystal violet cv assay, or incucyte ic. Overall, results from the crystal violet assay, clonogenic assay, celltiterfluor assay, alamar blue assay, and celltiterglo assay were comparable for most treatments.
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