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. In vitro toxicology assay kit resazurin based stock no. Resazurin dye has been broadly used as indicator of cell vi ability in several types of. Damaged and nonviable cells have lower innate metabolic activity, and generate a proportionally lower signal. Microplate alamar blue assay how is microplate alamar blue assay abbreviated. The incubation time of 24 to 30 h was chosen in order to not lose any potential positive extract because with longest. This is a trusted and established reagent which has been available since 1993.
Calculations assume 100 l final volume per well 96well plate. The susceptibility test conducted by the microplate alamar blue assay was using 96 well microtiter plate to evaluate the susceptibility of h37rv mtb reference strain to the extract. A fast and reproducible cell and 96well platebased method. Invitrogen alamarblue hs cell viability reagent life. 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. Living cells are metabolically active and are able to reduce via mitochondrial reductase, the nonfluorescent dye resazurin to the stronglyfluorescent dye resorufin fig. Resazurin, the active ingredient in the maba alamarblue biosource international, invitrogens biosource division, is. The resazurin assay also known as alamar blue assay offers a simple, rapid, and sensitive measurement for the viability of mammalian cells and bacteria. The disadvantage of this assay is that it is not a. For the invasion assay, the same filters were precoated with fibronectin. A simple colorimetric method to screen drug cytotoxicity against leishmania using the dye alamar blue. 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. Similarly, in the nccls planktonic assay 17, minimum bactericidal concentrations mbcs are obtained from the same wells as mics. Moreover, coban 2012 reported 100% agreement between the resazurin microplate assay for the rapid determination of mrsa and the cefoxitin mic.
Since the autoclaving procedure was not efficient in reducing the. Staphylococcus aureus biofilms were treated with eleven. This assay has excellent performance compared to other resazurinbased cell proliferation kits such as alamarblue, prestoblue, or celltiterblue. Microplate alamar blue assay for staphylococcus epidermidis biofilm susceptibility testing. A simple method to measure cell viability in proliferation and cytotoxicity assays 256 braz oral res 2009. 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. For the migration assay, we used 12 m millicel filters, with a 12 micro pore polycarbonate membrane. Moreover, coban 2012 reported 100% agreement between the resazurin microplate assay for the rapid determination of mrsa and the cefoxitin mic determined by the reference broth microdilution method. Alamarbluetm detects cell viability by utilizing a blue and nonfluorescent dye resazurin, which is converted to a pink and fluorescent dye resorufin. This colorimetric assay is based on the reduction of a yellow tetrazolium salt 34,5dimethylthiazol2yl2,5diphenyltetrazolium bromide or mtt to purple formazan crystals by metabolically active cells fig. Swirl the solution several times and pipette 100 l into the wells of a flat bottom microtiter plate. Resazurin, the active ingredient in the maba alamarblue biosource international, invitrogens biosource division, is permeable, blue, and virtually non.
It is a sensitive assay if working with higher than 5x10 3 cells per 100. Cell lines are often cultivated in contact with the target materials, and after a variable period. The results were compared to those of the proportion method pm on lj. Any multiwell microtiter plate reader equipped with a 595 nm filter. It is a nontoxic, water soluble, redoxsensitive dye that changes from its blue nonfluorescent state to a pinkhighlyfluorescent state upon reduction by viable cells. L collins and s g franzblau pharmacology research department, gillis w. A simple method to measure cell viability in proliferation. Cell counting kit8 product description cell counting kit8 is a colorimetric assay for the determination of viable cell numbers and can be used for cell proliferation assays as well as cytotoxicity assays. Overview alamarblue can be used in a wide range of scientific research areas and applications including experiments involving cell proliferation, cell viability, bioassays for relative cytotoxicity, cytokine assays, cell metabolism studies, drug susceptibility, and.
Equation 1 calculates percentage reduction of alamarblue using absorbance. 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. Compared to alamarblue, alamarblue hs contains highly purified resazurin and provides higher sensitivity, and a larger assay window. Find, read and cite all the research you need on researchgate. This results in colorimetric absorbance and fluorescence changes. A fast and reproducible cell and 96well platebased. Microplate alamar blue assay maba and low oxygen recovery. Maba, which is a highly sensitive, practical, and inexpensive method for assaying cell viability. I am doing alamar blue assay to test my drug cytotoxicity. 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. Franzblaumicroplate alamar blue assay versus bactec 460 system for highthroughput screening of compounds against mycobacterium tuberculosis and mycobacterium avium. The resazurin proved to be as suitable as alamar blue for determining the minimal inhibitory concentration of different antifungals against a.
Molecular probes alamarblue cell viability reagent 25ml. The optimal inoculum per well for a 96well micro plate was 2. Throughput in tuberculosis drug discovery was extremely limited prior to the introduction of microplate based susceptibility assays. It is a nontoxic, water soluble, redoxsensitive dye that changes from its bluenonfluorescent state to a pinkhighlyfluorescent state upon reduction by viable cells. Use of the bradford protein assay in a microtiter plate format.
In addition, two blank wells media only and two controlwells media plus alamarblue are defined on the plate. The mtt assay is used to measure cellular metabolic activity as an indicator of cell viability, proliferation and cytotoxicity. Long hansens disease center, baton rouge, louisiana 70894, usa. In vitro toxicology assay kit, resazurin based tox8. Since these assays are based on metabolic potentials such as mitochondrial enzyme activity in the target cells, it takes several hours to obtain results. Resazurin microtiter assay plate method for detection of. Pdf microplate alamar blue assay for paracoccidioides. Microplate alamar blue assay how is microplate alamar blue. Molecular probes alamarblue cell viability reagent life.
Resazurin cell viability assay offers a simple, rapid, reliable, sensitive, safe and costeffective measurement of cell viability. Obtain od 570od 600 for each sample if colorimetric detection method is chosen, or fluorescence signal from each sample deducted by background fluorescence from the background control, and plot a standard curve to identify the optimal cell concentration for your. Celltiterblue reagent is added directly to each well, the plates are incubated at 37c to allow cells to convert. Upon entering living cells, resazurin is reduced to resorufin, a compound that is red in color and highly fluorescent. Validation of the alamarblue assay as a fast screening.
Cellladen samples were sectioned, washed twice, and placed into a culture plate where 10% vv alamar blue was added to micro channels and incubated for 4 hours. Plate configuration provides for samples to be run in duplicate. Pdf microplate alamar blue assay for staphylococcus. A 96well plate containing the cells and the compounds to be tested is prepared using standard methods. Mar 16, 2012 to achieve this, we used a fluorescence reduction assay of alamar blue a resazurin in a microplate format microplate alamar blue assay. Franzblaumicroplate alamar blue assay versus bactec 460 system for highthroughput screening of compounds against mycobacterium tuberculosis and mycobacterium avium antimicrob. The basis for this assay is the reduction of copper in alkali by protein biuret reaction in the presence of the chromogenic agent bicinchoninic acid. Cell counting kit8 uses a tetrazolium salt, wst8, which produces the water soluble wst8 formazan. This rema plate method is very similar to the alamar blue assay, interpretation of results is very easy, and correlation with the pm was excellent. Application of a high throughput alamar blue biofilm.
Spectrophotometer plate reader capable of reading 570 600nm absorbance. Google scholar hansen j, bross p 2010 a cellular viability assay to monitor drug toxicity. L, relatively nontoxic and provided that it is carried out carefully, the same replicates can be followed over several time points. Pettit and christine a weber and melissa j kean and holger hoffmann and george r. Microplate alamar blue assay for staphylococcus epidermidis. Alamarblue assay for cell proliferation bmg labtech. Microplate alamar blue assay versus bactec 460 system for highthroughput screening of compounds against mycobacterium tuberculosis and mycobacterium avium. In regards to data reduction, a transformation that calculates reduction percentage using the molar extinction coefficients of reduced and oxidized alamarblue at 570. Resazurin is blue and nonfluorescent whereas resorufin is red and highly fluorescent. Description alamarbluetm fluorometric cell viability assay kit offers a simple, rapid, reliable, sensitive, safe and costeffective measurement of cell viability. Resazurin microtiter assay plate method for detection of susceptibility of multidrug resistant mycobacterium tuberculosis to secondline antituberculous drugs. The highly purified resazurin used for alamarblue hs reagent results in a 50% decrease in background fluorescence and a 100% increase in the signaltobackground ratio. A fluorometric indicator alamar blue, serotec of cell metabolic activity was utilized to determine the cell proliferation in the channels.
Throughput in tuberculosis drug discovery was extremely limited prior to the introduction of microplatebased susceptibility assays. Since it is also tested in liquid medium, it has not been implemented as a direct dst method due to the contamination problems that may arise. 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. Evaluation of alamar blue reduction for the in vitro assay of hepatocyte toxicity. Rapid, lowtechnology mic determination with clinical. Microplate alamar blue assay for susceptibility testing of. A colorimetric, microplate based alamar blue assay maba method was used to determine the mics of isoniazid inh, rifampin, streptomycin sm, and ethambutol emb for 34 peruvian mycobacterium tuberculosis isolates including both pansensitive and multidrugresistant strains and the h 37 rv strain by using bacterial suspensions prepared directly from solid media.
Microplate alamar blue assay how is microplate alamar. To achieve this, we used a fluorescence reduction assay of alamar blue a resazurin in a microplate format microplatealamarblueassay. The reagent allows the detection of microbial growth in microtiter plates without the use of spectrophotometer. Microtiter plate reader for reading absorbance at 570nm and 600nm.
The ab assay is a simple, onestep procedure, quite amenable to high throughput, whereby metabolic activity results in the chemical reduction of ab. Microtiter plates we use micro test iii flexible assay plate from falcon because they are cheap and work well. A 24well falcon plate was used as a feeder tray in which 600 l of culture medium was placed in each well. The ab minimum biofilm inhibitory concentration mbic was defined as the lowest drug concentration resulting in. Overview alamarblue can be used in a wide range of scientific research areas and applications including experiments involving cell proliferation, cell viability, bioassays for relative cytotoxicity, cytokine assays, cell metabolism studies, drug susceptibility, and toxicology studies simple and easy workflow just add the readytouse alamarblue solution to the cells, incubate for at least 1. This reaction produces a blue color with an absorbance peak at 562 nm.
Microplate alamar blue assay versus bactec 460 system for. The bioassay may also be used to establish relative cytotoxicity of agents within various chemical classes 3. Resazurin 7hydroxy3hphenoxazin3one 10oxide is a phenoxazine dye that is weakly fluorescent, nontoxic, cellpermeable, and redox. Singlestep, homogeneous, highthroughput cell quantitation. Pdf the use of the microplate alamar blue assay maba to.
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