Thermo scientific alamarblue cell viability assay reagent. A 96well plate containing the cells and the compounds to be tested is prepared using standard methods. Fluorescence left and absorbance right spectra of thermo scientific alamarblue reagent in oxidized and reduced states. Simply, the metabolic activity of cells converts soluble resazurin dye into fluorescent resorufin, and fluorescence excitation 535 nm, emission 590 nm of this dye was recorded. Damaged and nonviable cells have lower innate metabolic activity, and generate a proportionally lower signal. Only one appropriate substitute wavelength may be used. Because the indicator is a multicomponent solution, it is recommended that frozen alamarblue be warmed to 37c and shaken to ensure all components are completely in solution.
Measuring cytotoxicity or proliferation alamarblue assay. Alamarblue assay definition of alamarblue assay by. Validation of the alamarblue assay as a fast screening. The homogeneous, addandread assay format of the alamarblue cell viability reagent is fast, convenient, and readily amenable to automation and highthroughput assays.
Resazurin is blue and nonfluorescent whereas resorufin is red and highly fluorescent. Do not use the assay with media containing reagents with high redox potential e. Invitrogen alamarblue hs cell viability reagent 25 ml. Compared to alamarblue, alamarblue hs contains highly purified resazurin and provides higher sensitivity, and a larger assay window. 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. The resazurin assay protocol uses an indicator dye to measure oxidationreduction reactions which principally occur in the mitochondria of live cells. Format excitation emission general 540570 nm 580610 nm fluorescence.
The susceptibility pattern was clear at 60 min, so this time point was chosen as the endpoint for absorbance readings in all experiments. The spectral properties of celltiterblue reagent change upon reduction of resazurin to resoru. The ab minimum biofilm inhibitory concentration mbic was defined as the lowest drug concentration resulting in. 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.
Alamarblue cell viability reagent invitrogen, dal1025 is commonly used to assess cell health. Absorbance values may be affected by the type of plate whether round or flat bottom and the plate manufacturer. Microplate alamar blue assay versus bactec 460 system for highthroughput screening of compounds against mycobacterium tuberculosis and mycobacterium avium. This change can be detected using fluorescence or absorbance measurement figure 2 in datasheet. The wavelengths for maximal absorbance are 570 nm and 600 nm for the reduced and oxidized forms of alamarblue respectively. Fluorescence is more sensitive than absorbance and is the preferred detection method. Resazurin cell viability assay offers a simple, rapid, reliable, sensitive, safe and costeffective measurement of cell viability. 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. It is a nontoxic, water soluble, redoxsensitive dye that changes from its bluenonfluorescent state to a pinkhighlyfluorescent state upon reduction by viable cells. A simple method to measure cell viability in proliferation. Molecular probes alamarblue cell viability reagent.
Alamarblue cell viability assay reagent boster biological technology, pleasanton ca, usa, catalog. Alamarblue cell viability reagent from thermo fisher. How to perform a dualfluorescent aoeb assay video demonstration duration. Method for measuring cytotoxicity or proliferation using alamarblue by spectrophotometry. Cells can continue to grow and be subjected to alamarblue assay at a later time point. The ingredients have been optimized for use as a cell viability assay. The simultaneous alamar blue assay showed linear absorbance for both confluent and nonconfluent cell cultures. Monitor the absorbance of alamarblue at 570 nm, using 600 nm as a reference wavelength normalized to the 600 nm value. Harvest cells which are in the log phase of growth and determine cell count. I am doing alamar blue assay to test my drug cytotoxicity. The assay is based on detection of metabolic activity through an oxidationreduction redox indicator, which both fluoresces and changes colour in.
Mtt is a tetrazolium salt that is turned into a purple formazan product after reduction by mitochondrial enzymes that are only present in metabolically active live cells. The absorbance of test and control wells was read at 540 and 630 nm with a standard spectrophotometer. This assay has excellent performance compared to other resazurinbased cell proliferation kits such as alamarblue, prestoblue, or celltiterblue. Read fluorescence or absorbance signal is stable for 7 hours. 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 absorbance based instrumentation platforms, and. Important product information store the assay protected from light. The health of the cell can be monitored by the change in fluorescence andor absorbance. Nonviable cells cannot reduce the indicator dye and therefore do not generate a change in signal.
Microplate alamar blue assay for staphylococcus epidermidis. Upon entering living cells, resazurin is reduced to resorufin, a compound that is red in color and highly fluorescent. Microtiter plate reader for reading absorbance at 570nm and 600nm. Dec 05, 2010 how to perform a dualfluorescent aoeb assay video demonstration duration. Adjust the cell count to 1 x 10 4 cellsml suggested cell density. Resazurin 7hydroxy3hphenoxazin3one 10oxide is a phenoxazine dye that is weakly fluorescent, nontoxic, cellpermeable, and redox. The mtt assay is used to measure cellular metabolic activity as an indicator of cell viability, proliferation and cytotoxicity. Schematic representative of alamarblue cell viability reagent undergoing reduction within the cells figure 2. Measuring cytotoxicity or proliferation alamarblue assay protocol. Resazurin is dark blue in color and has little intrinsic. Absorbance 20 minutes 2 hours room temperature incubation 10 minutes 2 hours low cell number absorbance. Alamar blue was then added, and absorbance was determined 30 to 90 min after the addition of ab fig. Isolation of alamar blue in order to obtain a definitive identification of alamar blue, the dye present in the reagent was isolated using solid. Measure absorbance of the samples collected in the previous steps at wavelengths of 570 nm and 600 nm using a plate.
This results in colorimetric absorbance and fluorescence changes. In conclusion, the alamar blue assay was shown to be an excellent tool to determine drug sensitivities of the human pathogenic african trypanosomes, t. Resazurin cell viability kit protocol specific for. 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 fluorescence or absorbance detector. Alamarblue assay definition of alamarblue assay by medical. 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.
This change can be detected using fluorescence or absorbance measurement figure 2. Measure absorbance or fluorescence using a plate reader. Alamarblue assay for cell proliferation bmg labtech. The optimum cell density may vary between cell types. L, relatively nontoxic and provided that it is carried out carefully, the same replicates can be followed over. The absorbance spectrum for reduced and oxidized forms of the resazurin dye are highlighted in figure 1. Microplate alamar blue assay for susceptibility testing of. Can anyone explain about alamar blue assay calculation. 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. Application of a high throughput alamar blue biofilm.
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 absorbance based instrumentation platforms, and. Material amount concentration storage stability alamarblue reagent 25ml cat. A du7000 spectrophotometer beckman was used to measure the absorbance of alamar blue, resazurin and resorufin. Fluorescence and absorbance spectra of alamarblue reagent in oxidized and reduced. Fluorescence can be read using 544 nm excitation and 590 nm emission wavelengths, or absorbance can be read using a. Alamar blue ab is a watersoluble dye that has been previously used for quantifying in vitro viability of various cells fields and lancaster, 1993. O1 molar extinction coefficient e of oxidized alamarblue blue at 570 nm o2 e of oxidized alamarblue at 600 nm a1 absorbance of test wells at 570 nm a2 absorbance of test wells at 600 nm p1 absorbance of positive growth control well cells plus alamarblue but no test agent 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 of alamarblue reagent, is a nontoxic, cellpermeable compound that is blue in color and virtually nonfluorescent. Isolation of alamar blue in order to obtain a definitive identification of alamar blue, the dye present in the reagent was isolated using solidphase. Thaw out resazurin solution if kept frozen and warm it to 37c to ensure all components are completely in solution. Monitor cell proliferation and cell viability for in vitro cytotoxicity studies e.
In this study alamar blue demonstrates several advantages over 3h thymidine. P2 absorbance of positive growth control well cells plus alamarblue but no test agent at 600 nm. Alamarblue cell viability assay for 3d cell culture. The color change and fluorescence increase resulting from cell viability changes allow the detection of alamarblue reagent by either an absorbance or fluorescencebased. A simple method to measure cell viability in proliferation and cytotoxicity assays abstract. It is a sensitive assay if working with higher than 5x10 3 cells per 100. Investigation of the alamar blue resazurin fluorescent. The alamarblue dye is a redox indicator that yields a colorimetric change and a fluorescent signal in response to metabolic activity.
Upon entering a living cell, prestoblue reagent is reduced to resorufin which is red in color and highly fluorescent. Metabolically active cells continuously convert the prestoblue reagent. 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 bioassay may also be used to establish relative cytotoxicity of agents within various chemical classes 3. When incubated with viable cells, the reagent changes color from blue to red and becomes fluorescent figure 1 in datasheet. 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.
Whereas other commercially available resazurin solutions such as. The mtt cell viability assay kit provides a simple method for determining live cell numbers by absorbance on a microplate reader. Apr 03, 2020 replace the media in your cell culture. C protect from light when stored as directed this kit is stable until the expiration date printed on the product. By monitoring the absorbance at 570 nm and 600 nm, relative metabolic activity for the cells can be determined. Isolation of alamar blue in order to obtain a definitive identification of alamar blue, the dye present in. Because the wavelengths overlap it is necessary to measure the absorbance at both wavelengths. What does the data i have retrieved from alamar blue assay.
Multiple applications of alamar blue as an indicator of. Absorbance is monitored at 570 nm and 600 nm fluorescence is monitored at 530560 nm excitation wavelength and 590 nm emission wavelength alamarblue reduction is regularly measured using absorbance, which gives good levels of accuracy for most experiments, and is particularly easy to use. 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. Investigation of the alamar blue resazurin fluorescent dye. Whether you perform cell viability assays in a single plate or process hundreds of plates at a. This protocol takes you stepbystep through the use of alamarblue reagent to monitor viability in mammalian cells using a fluorescence. The fluorescence output is proportional to the number of viable. In general more sensitive readings can be obtained using fluorescence, especially when attempting to measure very small changes in reduction. It is a proven safe and nontoxic dye used for quantitative analysis of cell viability and cell proliferation, for.
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