Measuring cytotoxicity or proliferation alamarblue assay protocol. The alamarblue dye is a redox indicator that yields a colorimetric change and a fluorescent signal in response to metabolic activity. Fluorescence can be read using 544 nm excitation and 590 nm emission wavelengths, or absorbance can be read using a. The mtt assay is used to measure cellular metabolic activity as an indicator of cell viability, proliferation and cytotoxicity. L, relatively nontoxic and provided that it is carried out carefully, the same replicates can be followed over. Living cells are metabolically active and are able to reduce via mitochondrial reductase, the nonfluorescent dye resazurin to the stronglyfluorescent dye resorufin fig. Apr 03, 2020 replace the media in your cell culture. It is a nontoxic, water soluble, redoxsensitive dye that changes from its bluenonfluorescent state to a pinkhighlyfluorescent state upon reduction by viable cells. The ingredients have been optimized for use as a cell viability assay. The homogeneous, addandread assay format of the alamarblue cell viability reagent is fast, convenient, and readily amenable to automation and highthroughput assays.
Alamar blue ab is a watersoluble dye that has been previously used for quantifying in vitro viability of various cells fields and lancaster, 1993. Absorbance 20 minutes 2 hours room temperature incubation 10 minutes 2 hours low cell number absorbance. Alamarblue assay definition of alamarblue assay by medical. Alamarblue assay definition of alamarblue assay by. The simultaneous alamar blue assay showed linear absorbance for both confluent and nonconfluent cell cultures. The assay is based on detection of metabolic activity through an oxidationreduction redox indicator, which both fluoresces and changes colour in. Alamarblue cell viability assay for 3d cell culture. Resazurin 7hydroxy3hphenoxazin3one 10oxide is a phenoxazine dye that is weakly fluorescent, nontoxic, cellpermeable, and redox. 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. Measuring cytotoxicity or proliferation alamarblue assay. 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.
Monitor the absorbance of alamarblue at 570 nm, using 600 nm as a reference wavelength normalized to the 600 nm value. Alamar blue was then added, and absorbance was determined 30 to 90 min after the addition of ab fig. A simple method to measure cell viability in proliferation and cytotoxicity assays abstract. A 96well plate containing the cells and the compounds to be tested is prepared using standard methods. The health of the cell can be monitored by the change in fluorescence andor absorbance. Can anyone explain about alamar blue assay calculation. Measure absorbance or fluorescence using a plate reader.
Application of a high throughput alamar blue biofilm. The bioassay may also be used to establish relative cytotoxicity of agents within various chemical classes 3. 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. Investigation of the alamar blue resazurin fluorescent. The spectral properties of celltiterblue reagent change upon reduction of resazurin to resoru. Schematic representative of alamarblue cell viability reagent undergoing reduction within the cells figure 2. 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. Microplate alamar blue assay versus bactec 460 system for highthroughput screening of compounds against mycobacterium tuberculosis and mycobacterium avium. Method for measuring cytotoxicity or proliferation using alamarblue by spectrophotometry. This protocol takes you stepbystep through the use of alamarblue reagent to monitor viability in mammalian cells using a fluorescence. What does the data i have retrieved from alamar blue assay.
Important product information store the assay protected from light. Harvest cells which are in the log phase of growth and determine cell count. Cells can continue to grow and be subjected to alamarblue assay at a later time point. By monitoring the absorbance at 570 nm and 600 nm, relative metabolic activity for the cells can be determined. Do not use the assay with media containing reagents with high redox potential e. Fluorescence and absorbance spectra of alamarblue reagent in oxidized and reduced. Material amount concentration storage stability alamarblue reagent 25ml cat. Only one appropriate substitute wavelength may be used. 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.
Validation of the alamarblue assay as a fast screening. It is a sensitive assay if working with higher than 5x10 3 cells per 100. Adjust the cell count to 1 x 10 4 cellsml suggested cell density. The fluorescence output is proportional to the number of viable. 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. The absorbance spectrum for reduced and oxidized forms of the resazurin dye are highlighted in figure 1. 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.
Dec 05, 2010 how to perform a dualfluorescent aoeb assay video demonstration duration. 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. When incubated with viable cells, the reagent changes color from blue to red and becomes fluorescent figure 1 in datasheet. The resazurin assay also known as alamar blue assay offers a simple, rapid, and sensitive measurement for the viability of mammalian cells and bacteria. Because the wavelengths overlap it is necessary to measure the absorbance at both wavelengths. The optimum cell density may vary between cell types. 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. Microplate alamar blue assay for staphylococcus epidermidis. Isolation of alamar blue in order to obtain a definitive identification of alamar blue, the dye present in the reagent was isolated using solidphase. Alamarblue cell viability reagent invitrogen, dal1025 is commonly used to assess cell health.
The resazurin assay protocol uses an indicator dye to measure oxidationreduction reactions which principally occur in the mitochondria of live cells. 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. Absorbance values may be affected by the type of plate whether round or flat bottom and the plate manufacturer. 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. Plate cells and expose to test agent as determined by. The ab minimum biofilm inhibitory concentration mbic was defined as the lowest drug concentration resulting in. Resazurin cell viability assay offers a simple, rapid, reliable, sensitive, safe and costeffective measurement of cell viability. This change can be detected using fluorescence or absorbance measurement figure 2. In this study alamar blue demonstrates several advantages over 3h thymidine.
Alamarblue cell viability assay reagent boster biological technology, pleasanton ca, usa, catalog. Resazurin is dark blue in color and has little intrinsic. Whether you perform cell viability assays in a single plate or process hundreds of plates at a. This change can be detected using fluorescence or absorbance measurement figure 2 in datasheet. It is a proven safe and nontoxic dye used for quantitative analysis of cell viability and cell proliferation, for. Molecular probes alamarblue cell viability reagent. P2 absorbance of positive growth control well cells plus alamarblue but no test agent at 600 nm. 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. How to perform a dualfluorescent aoeb assay video demonstration duration. C protect from light when stored as directed this kit is stable until the expiration date printed on the product. Resazurin, the active ingredient of alamarblue reagent, is a nontoxic, cellpermeable compound that is blue in color and virtually nonfluorescent. I am doing alamar blue assay to test my drug cytotoxicity. Format excitation emission general 540570 nm 580610 nm fluorescence.
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. Damaged and nonviable cells have lower innate metabolic activity, and generate a proportionally lower signal. 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. In conclusion, the alamar blue assay was shown to be an excellent tool to determine drug sensitivities of the human pathogenic african trypanosomes, t. A simple method to measure cell viability in proliferation. Microtiter plate reader for reading absorbance at 570nm and 600nm.
Monitor cell proliferation and cell viability for in vitro cytotoxicity studies e. The wavelengths for maximal absorbance are 570 nm and 600 nm for the reduced and oxidized forms of alamarblue respectively. Upon entering a living cell, prestoblue reagent is reduced to resorufin which is red in color and highly fluorescent. In general more sensitive readings can be obtained using fluorescence, especially when attempting to measure very small changes in reduction. Metabolically active cells continuously convert the prestoblue reagent. This results in colorimetric absorbance and fluorescence changes. Alamarblue assay for cell proliferation bmg labtech. Microplate alamar blue assay for susceptibility testing of. Read fluorescence or absorbance signal is stable for 7 hours. 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.
Thaw out resazurin solution if kept frozen and warm it to 37c to ensure all components are completely in solution. A du7000 spectrophotometer beckman was used to measure the absorbance of alamar blue, resazurin and resorufin. Whereas other commercially available resazurin solutions such as. Isolation of alamar blue in order to obtain a definitive identification of alamar blue, the dye present in. 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. 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. Resazurin cell viability kit protocol specific for. When fluorescence instrumentation is unavailable, monitor the absorbance of alamarblue reagent. Fluorescence is more sensitive than absorbance and is the preferred detection method. Upon entering living cells, resazurin is reduced to resorufin, a compound that is red in color and highly fluorescent.
The susceptibility pattern was clear at 60 min, so this time point was chosen as the endpoint for absorbance readings in all experiments. The mtt cell viability assay kit provides a simple method for determining live cell numbers by absorbance on a microplate reader. This assay has excellent performance compared to other resazurinbased cell proliferation kits such as alamarblue, prestoblue, or celltiterblue. Resazurin is blue and nonfluorescent whereas resorufin is red and highly fluorescent. 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. The color change and fluorescence increase resulting from cell viability changes allow the detection of alamarblue reagent by either an absorbance or fluorescencebased. Alamarblue cell viability reagent from thermo fisher. 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. Fluorescence left and absorbance right spectra of thermo scientific alamarblue reagent in oxidized and reduced states.
Investigation of the alamar blue resazurin fluorescent dye. The alamar blue assay to determine drug sensitivity of. Multiple applications of alamar blue as an indicator of. Nonviable cells cannot reduce the indicator dye and therefore do not generate a change in signal. No difference in ic 50 values was obtained employing the alamar blue assay either with fluorescence or absorbance measurement. Thermo scientific alamarblue cell viability assay reagent. The absorbance of test and control wells was read at 540 and 630 nm with a standard spectrophotometer.