Alamar blue reduction arrayscan microscopy reporter gene activation spectrophotometry radioactivity hplc and hpec elisa readout type single multiplexed multiparametric assay provider acea apredica attagene bioreliance bioseek ceetox cellzdirect tox21ncats nheerl mesc nheerl zebrafish novascreenperkin elmer odyssey thera vala sciences assay. Pdf the alamarblue assay is based on fluorometric detection of metabolic mitochondrial activity of cells. Convenient addandread formatno mixing, no washing, no cell lysis compatible with either fluorescence or absorbancebased instrumentation. 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. 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.
A variety of parameters for the ab assay were standardized at the outset using s. Cell viability assays such as cell titer blue and alamar blue rely on the reducing property of viable cells to reduce the reagent dye to a product which gives a. Analysis of cell viability by the alamarblue assay. Section 3 hazards identification this product is not classified as hazardous and no known health hazard is known to be associated with exposure to this product. When 10 5 cfu of the mesophilic bacteria were placed in a well, the time for converting the color required from 2 to 4 h at 37 c incubation.
Resazurin has been identified as the main component of alamar blue 22. Ldh cytotoxicity assay, caspase 37 assay, mttmts assay. At various time intervals, the redox reaction, in which ab is reduced by the cells, was measured by absorbance readings at 540 and 630 nm. Optimization of a rapid viability assay for mycobacterium. Multiple applications of alamar blue as an indicator of metabolic. Factors to consider for designing and optimizing assays.
This assay has excellent performance compared to other resazurinbased cell proliferation kits such as alamarblue, prestoblue, or celltiterblue. The medium 7h9s without inoculum control well was used to decide the interference of 7h9s to alamar blue. Imberhorne lane, east grinstead, west sussex rh19 1qx england z tel 44 0 42. The spectral properties of celltiterblue reagent change upon reduction of resazurin to resoru. Pdf multiple applications of alamar blue as an indicator of. Microplate alamar blue assay versus bactec 460 system for highthroughput screening of compounds against mycobacterium tuberculosis and mycobacterium avium. Assessment of cell proliferation with resazurinbased. 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. Disadvantages of metabolic reduction assays drug discovery.
Alamar blue dye is a fluorogenic redox indicator that is converted from the oxidized form to the. Original article resazurin microtiter assay for detection of. Just dilute into your cell growth media and place on your cells. Glucocorticoids and lithium reciprocally regulate the. Microplate alamar blue assay for staphylococcus epidermidis. Alamar blue monitors the reducing environment of the living cell. Staphylococcus aureus biofilms were treated with eleven. Alamar blue ab is a watersoluble dye that has been previously used for quantifying in vitro viability of various cells fields and lancaster, 1993. The alamarblue dye is a redox indicator that yields a colorimetric change and a fluorescent signal in response to metabolic activity.
A microtiter alamarblue assay was adapted and optimized for mycobacterium avium subsp. In alamarblue assay the growing cells cause a chemical reduction of the alamarblue dye from nonfluorescent blue to red fluorescent. Resazurin, the active ingredient of alamarblue reagent, is a nontoxic, cellpermeable compound that is blue in color and virtually nonfluorescent. Cell viability assays such as cell titer blue and alamar blue rely on the reducing property of viable cells to reduce the reagent dye to a product which gives a fluorescent signal.
Bioteks visual abstracts are brief, animated presentations that describe the workflow of a single specific application. Throughput in tuberculosis drug discovery was extremely limited prior to the introduction of microplatebased susceptibility assays. Microtitre panel containing a range of antifungal drugs and alamar blue. Specifically, the system incorporates an oxidationreduction redox indicator that both fluoresces and changes color in response to chemical reduction of growth medium resulting from cell growth. Validation of the alamarblue assay as a fast screening. Alamar blue ab was diluted at 10% in media and then added to the well 100. Suitable for colorimetric or fluorometric detection. The assay is comparable with respect to known reference compound activities as those reported in the literature for the alamar blue assay in format 11, as well as the reported luciferase based assay in a 96well format.
Therefore, a lot of research on cell viability assay has been carried out. Celltiterblue cell viability assay technical bulletin tb317. Alamarblue cell viability reagent from thermo fisher scientific. Blue and weakly fluorescent resorufin red and highly fluorescent the assay incorporates a reductionoxidation redox indicator that both fluoresces and undergoes colorimetric change in response to cellular metabolic reduction. 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.
The absorbance spectrum for reduced and oxidized forms of the resazurin dye are highlighted in figure 1. The alamar blue assay provides accurate timecourse measurements, has high sensitivity and linearity, involves no cell lysis, is ideal for use with postmeasurement functional assays, is flexible as it can be used with different cell models, is scalable and can be used with fluorescence andor absorbancebased instrumentation platforms, and. Upon entering living cells, resazurin is reduced to resorufin, a compound that is red in color and highly fluorescent. Rifampin rifampicin was used to demonstrate that this method has applications for highthroughput screening against. Singlestep, homogeneous, highthroughput cell quantitation. Performing viability assays on primary cells using trypan blue and aopi. By monitoring the absorbance at 570 nm and 600 nm, relative metabolic activity for the cells can be determined. Multiple applications of alamar blue as an indicator of. Calculations assume 100 l final volume per well 96well plate. Although these initial experiments typically involve a large number of data points, they are the celltiterblue cell viability assay.
Pdf evaluation of the alamarblue assay for adherent cell. Performing viability assays on primary cells using trypan blue and aopi duration. Bioactivity profiling using primary human cell systems in. Resazurin is dark blue in color and has little intrinsic fluorescence. 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.
Application of a high throughput alamar blue biofilm. A luciferase based viability assay for atp detection in 384. Nov 12, 2008 alamar blue assay is a rapid and simple nonradioactive assay to measure the number of cells. 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. Original article in vitro interactions between r207910 and. Sep 10, 2012 the alamar blue assay provides accurate timecourse measurements, has high sensitivity and linearity, involves no cell lysis, is ideal for use with postmeasurement functional assays, is flexible as it can be used with different cell models, is scalable and can be used with fluorescence andor absorbancebased instrumentation platforms, and.
The parameters that were explored and optimised to provide a hts assay in 384well format are discussed in detail. The ingredients have been optimized for use as a cell viability assay. The assay is based on detection of metabolic activity through an oxidationreduction redox indicator, which both fluoresces and changes colour in. Colorimetric alamarblue assay as a bacterial concentration. Alamarblue assay definition of alamarblue assay by medical. This product is however used in conjunction with fungal cultures and any hazards associated. Among many evaluation methods of cell viability, the alamar blue method is widely accepted for its simple operation and. Among many evaluation methods of cell viability, the alamar blue method is widely accepted for its. To assay for viability, simply add the premixed alamarblue reagent to cells in. The amount of fluorescence produced is proportional to the number of living cells. Table 3 ab presents the absorbance values for the oxidized and reduced forms of the indicator in several commonly used culture media. Using cell concentrations ranging from 104 to 108 cfuml, a minimum incubation time to indicate viability was obtained after 24 h.
Resazurin is dark blue in color and has little intrinsic. Sensitivity of mtt assay limits signal to background ratio 1 bonnier et al. Resazurin 7hydroxy3hphenoxazin3one 10oxide is a phenoxazine dye that is weakly fluorescent, nontoxic, cellpermeable, and redox. In vitro assay of staphylococcus aureus enterotoxin a. Analysis of cell viability by the alamarblue assay request pdf. A 96well plate containing the cells and the compounds to be tested is prepared using standard methods. Cytotoxicity assay for hts in 96 well plates with alamar blue. Analysis of cell viability by the alamarblue assay csh protocols. The bioassay may also be used to establish relative cytotoxicity of agents within various chemical classes 3. It is a nontoxic, water soluble, redoxsensitive dye that changes from its bluenonfluorescent state to a pinkhighlyfluorescent state upon reduction by viable cells. The goal was to make the biofilm susceptibility assay as similar to the nccls planktonic susceptibility assay 17 as possible. 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. You will have to determine the length of time for development though.
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. Comparison of alamar blue and mtt assays for high throughput. Original article resazurin microtiter assay for detection. The rema was very similar to the alamar blue assay and the correlation with the pm was perfect 4, 1921. A flow diagram summarizing the celltiterblue assay protocol is shown in figure 3. 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. Dont let the color get really pink or you will have saturated the assay. The attached pdf talks of proscons of different assays.
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