Measuring cytotoxicity or proliferation alamarblue assay protocol. Alamarblue assay definition of alamarblue assay by. 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 use of alamar blue assay for quantitative analysis of. Resazurin 7hydroxy3hphenoxazin3one 10oxide is a phenoxazine dye that is weakly fluorescent, nontoxic, cellpermeable, and redox. A fluorometric indicator alamar blue, serotec of cell metabolic activity was utilized to determine the cell proliferation in the channels. 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 internal environment of proliferating cells is more reduced than that of nonproliferating cells. Cytotoxicity against human jurkattall cells after 48 hrs by alamar blue assay.
Section 5 fire fighting measures foam, dry powder, or carbon dioxide fire extinguisher as appropriate to local fire conditions. The alamarblue dye is a redox indicator that yields a colorimetric change and a fluorescent signal in response to metabolic activity. The assay is based on detection of metabolic activity through an oxidationreduction redox indicator, which both fluoresces and changes colour in. The alamarblue assay is based on fluorometric detection of metabolic mitochondrial activity of cells. The fluorescence output is proportional to the number of viable.
Staphylococcus aureus biofilms were treated with eleven. Compared to alamarblue, alamarblue hs contains highly purified resazurin and provides higher sensitivity, and a larger assay window. The broth was incubated at 36 o c and readings taken of 1 ml samples every 30 min, absorbance was measured at 560 nm and 605 nm. Assessment of cell proliferation with resazurinbased. In this study, we determined the methodology for application of the assay to radiation response experiments in 96well plates. Therefore, a lot of research on cell viability assay has been carried out. The use of alamar blue assay for quantitative analysis of viability, migration and invasion of choriocarcinoma cells. If however the spilled alamar blue has been in contact with a culture the spill should be. A colorimetric, microplatebased 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 h37rv strain by using bacterial suspensions prepared directly from solid media. Section 6 accidental release measures in general the affected area should be treated as any other non hazardous liquid spill. By monitoring the absorbance at 570 nm and 600 nm, relative metabolic activity for the cells can be determined. 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. The absorbance spectrum for reduced and oxidized forms of the resazurin dye are highlighted in figure 1. Singlestep, homogeneous, highthroughput cell quantitation.
A 96well plate containing the cells and the compounds to be tested is prepared using standard methods. Aid 753934 cytotoxicity against human jurkattall cells. Fluorescence can be read using 544 nm excitation and 590 nm emission wavelengths, or absorbance can be read using a. Format recommended incubation time bottomread fluorescence 10 minutes 2 hours topread fluorescence 30 minutes 2 hours absorbance 20 minutes 2 hours room temperature incubation 10 minutes 2 hours. The cell proliferation assay reagent alamarblue is designed to provide a rapid and sensitive measure of cell proliferation and cytotoxicity in various human and animal cell lines, bacteria and fungi. Alamar blue is also sensitive, capable of detecting as few as 100 cells in a well of a microtiter plate. Microplate alamar blue assay for susceptibility testing of. 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. Compounds such as tetrazolium salts and alamarblue, which can be reduced by cellular metabolic intermediates, can be used to monitor cellular proliferation. Rapid, lowtechnology mic determination with clinical.
Microplate alamar blue assay versus bactec 460 system for highthroughput screening of compounds against mycobacterium tuberculosis and mycobacterium avium. In vitro toxicology assay kit resazurin based stock no. Alamarblue cell viability reagent from thermo fisher scientific. The attached pdf talks of proscons of different assays. Alamarblue cell viability reagent from thermo fisher. The tetrazolium salts and alamar blue redox dyes can be quantified with a range of instruments for conventional or highthroughput studies using, for example, standard spectrophotometers or spectrofluorometers or plate readers for. The ab minimum biofilm inhibitory concentration mbic was defined as the lowest drug concentration resulting in. Pdf the use of alamar blue assay for quantitative analysis of. Covid19 is an emerging, rapidly evolving situation. The bioassay may also be used to establish relative cytotoxicity of agents within various chemical classes 3. Validation of the alamarblue assay as a fast screening. Multiple applications of alamar blue as an indicator of. Resazurin cell viability assay offers a simple, rapid, reliable, sensitive, safe and costeffective measurement of cell viability.
Alamar blue assay is a rapid and simple nonradioactive assay to measure the number of cells. Trek diagnostic systems, in comparison with these other methods of susceptibility testing, may have many potential advantages. Glucocorticoids and lithium reciprocally regulate the. Resazurin, the active ingredient of alamarblue reagent, is a nontoxic, cellpermeable compound that is blue in color and virtually nonfluorescent. The celltiterblue assay is based on the ability of living cells to convert a redox dye resazurin into a fluorescent end product resorufin. Comparison of flow cytometric and alamar blue tests with the. Example data test wells contain 10% alamarblue in 100. The intersample and intrasample variability of the results obtained with the microplate alamar blue assay for the indirect drug susceptibility testing of mycobacterium tuberculosis was investigated.
Among many evaluation methods of cell viability, the alamar blue method is widely accepted for its simple operation and. Whether you perform cell viability assays in a single plate or process hundreds of plates at a. Evaluation of the alamarblue assay for adherent cell. The specific advantages of the alamar blue assay over the 3 hthymidine assay for lymphocyte proliferation studies are. Microplate alamar blue assay maba and low oxygen recovery. 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. The simple protocol involves adding a single reagent directly to cells cultured in serumsupplemented medium. Safety data sheet section 1 chemical name and company details. Whether you perform cell viability assays in a single plate or process hundreds of plates at a time, the readytouse reagent is designed for fast and easy. The alamarblue assay was used to assess cell viability serotec, oxon, uk page et al. It can be applied in studies concentrating on animal, plant, yeast, and bacteria cells. The inexpensive microplate alamar blue assay maba is an indirect colorimetric dst method for determining the mics of tb drugs for strains of mycobacterium tuberculosis 10. Application of a high throughput alamar blue biofilm. 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 reduction of alamarblue results in a measurable color shift. Drugs with antioxidant properties can interfere with cell. Alamar blue dye is a fluorogenic redox indicator that is converted from the oxidized form to the. Calculations assume 100 l final volume per well 96well plate. It is a proven safe and nontoxic dye used for quantitative analysis of cell viability and cell proliferation, for. Alamar blue protocol nov302006 has any one done the cell viability cell toxicity assay using alamar blue. The 100% reduced form of alamarblue was produced by autoclaving controls ie. Upon entering living cells, resazurin is reduced to resorufin, a compound that is red in color and highly fluorescent. When compared with gold standards such as bactec 460. This assay has excellent performance compared to other resazurinbased cell proliferation kits such as alamarblue, prestoblue, or celltiterblue.
Table 3 ab presents the absorbance values for the oxidized and reduced forms of the indicator in several commonly used culture media. General method for measuring cytotoxicity or proliferation using alamarblue. 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. This product is however used in conjunction with fungal cultures and any hazards associated. Analysis of cell viability by the alamarblue assay request pdf. Inter and intraassay reproducibility of microplate alamar. Nov 12, 2008 alamar blue assay is a rapid and simple nonradioactive assay to measure the number of cells. It is a nontoxic, water soluble, redoxsensitive dye that changes from its bluenonfluorescent state to a pinkhighlyfluorescent state upon reduction by viable cells. Living cells are metabolically active and are able to reduce via mitochondrial reductase, the nonfluorescent dye resazurin to the stronglyfluorescent dye resorufin fig. Cellladen samples were sectioned, washed twice, and placed into a culture plate where 10% vv alamar blue was added to microchannels and incubated for 4 hours. We optimized the original protocol of alamarblue assay that usually. 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. The protocol with the reagents is not very clear and cites references.
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. 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. Increase of incubation period to 24 h at 37 c protected from light. 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. In this assay, the redox indicator alamar blue turns from blue to pink in the presence of mycobacterial growth. The performance of flow cytometry and the microplate alamar blue assay in determining susceptibility of mycobacterium tuberculosis was assessed by testing 150 brazilian isolates. This protocol describes viability measurements for cell cultures in a well tissue culture plate using alamarblue resazurin. Pdf optimized alamarblue assay protocol for drug doseresponse. Comparison of flow cytometric and alamar blue tests with.
So one ends up going round and round without any clear conclusion. Alamarblue assay definition of alamarblue assay by medical. In vitro toxicology assay kit, resazurin based tox8 bulletin. 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. In vitro toxicology assay kit, resazurin based tox8. Throughput in tuberculosis drug discovery was extremely limited prior to the introduction of microplatebased susceptibility 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. 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. Analysis of cell viability by the alamarblue assay. Inter and intraassay reproducibility of microplate. Common assays used to test cellular viability include the mtt and mts assays, which.
1399 170 1177 1219 772 1298 1127 250 1282 1053 1511 871 995 1460 1155 182 1032 1505 1282 106 1089 987 79 1154 1368 1485 705 4 738 942 643 1157 445 110 341 1505 1426 1202 990 1057 1493 498 1209 444 571