Hepg2 cells were incubated with alamar blue as described in materials and methods. 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. 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. Standardized method for in vitro antifungal susceptibility testing of candida albicans biofilms. Pdf cell viability assessment using the alamar blue. Resazurin cell viability assay offers a simple, rapid, reliable, sensitive, safe and costeffective measurement of cell viability. A microplate version of the alamar blue assay maba with modi. Molecular probes alamarblue cell viability reagent 25ml. These additives also slow the rate of generation of the fluorescent product. Resazurin is blue and nonfluorescent whereas resorufin. Rapid, lowtechnology mic determination with clinical. 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. Data may be collected using either fluorescencebased or absorbancebased.
Analysis of cell viability by the alamarblue assay priti. When incubated withviable cells, the reagent changes color from blue to red and becomes fluorescent figure 1. 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. Assessment of cell proliferation with resazurinbased. In addition, two blank wells media only and two controlwells media plus alamarblue are defined on the plate. We would like to show you a description here but the site wont allow us. Analysis of cell viability by the alamarblue assay cold spring. 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. The alamarblue resazurin dye in its oxidized form is blue in color and nonfluorescent. Compared to alamarblue, alamarblue hs contains highly purified resazurin and provides higher sensitivity, and a larger assay window. The ingredients have been optimized for use as a cell viability assay. Alamar blue monitors the reducing environment of the living cell. For both panels, values were deducted from the autofluorescence of alamar blue without cells. Among many evaluation methods of cell viability, the alamar blue method is widely accepted for its simple operation and.
For the measurement of metabolic activity and proliferation of living cells. 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. In vitro antimycobacterial activity of six cameroonian. Plate configuration provides for samples to be run in duplicate. Microplate alamar blue assay for susceptibility testing of. Multiple applications of alamar blue as an indicator of. Therefore, a lot of research on cell viability assay has been carried out. For example, ab results can be read visually, spectrofluorometrically or spectrophotometrically, its reduction is dependent on active metabolism, it is amenable to highthroughput, its use. Resazurin powder,bioreagent,cellculture 627588 sigma. Alamar blue fluorescence as a function of cell number. Analysis of cell viability by the alamarblue assay. By monitoring the absorbance at 570 nm and 600 nm, relative metabolic activity for the cells can be determined.
Measurement of cell proliferation in response to growth factors, cytokines and nutrients. Microtiter plate reader for reading absorbance at 570nm and 600nm. The resazurin assay also known as alamar blue assay offers a simple, rapid, and sensitive measurement for the viability of mammalian cells and bacteria. Quantitation of jurkat cells using the cellquant alamarblue cell viability reagent.
It uses the indicator dye resazurin to measure the. This change can be detected using fluorescence or absorbance measurement. Multiple applications of alamar blue as an indicator of metabolic. A fluorometric indicator alamar blue, serotec of cell metabolic activity was utilized to determine the cell proliferation in the channels. Comparisons of 2d and 3d cell culture models in literature have indicated differences in cellular morphology and metabolism, commonly attributed the better representation of in vivo conditions of the latter cell culture environment. In this paper, we describe the application of alamarblue, a new and versatile metabolic dye, for the. The fluorescence output is proportional to the number of viable. The spectral properties of celltiterblue reagent change upon reduction of resazurin to resoru. Following culture for days 1, 3 and 7, the basic medium was replaced with a medium. Alamarblue assay definition of alamarblue assay by medical.
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. Trek diagnostic systems, in comparison with these other methods of susceptibility testing, may have many potential advantages. The alamarblue test was performed to evaluate the cell proliferation. This can be measured as absorbance or fluorescence.
Drugs with antioxidant properties can interfere with cell. Jul 23, 2018 in summary, precise and reliable analysis of cell viability and proliferation for 3d cell cultures remains a challenging task. 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. It is a quantitative assay that allows rapid and convenient handling of a high number of samples. Sep 10, 2012 these findings suggest that the alamar blue assay may not be appropriate for studying proliferation in tendonderived cells. Resazurin sodium salt has been used in viability assay. In summary, precise and reliable analysis of cell viability and proliferation for 3d cell cultures remains a challenging task. I am doing alamar blue assay to test my drug cytotoxicity. Consequently, the alamarblue assay requires longer incubation times compared to resazurin. To take the place of a standard livedead assay, resazurin also be multiplexed with chemiluminescent assays, such as cytokine assays, caspase assays to measure apoptosis, or reporter assays to measure a gene or a protein expression. The assay is based on detection of metabolic activity through an oxidationreduction redox indicator, which both fluoresces and changes colour in. The simple protocol involves adding a single reagent directly to cells cultured in serumsupplemented medium. 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 irreversible reaction of resazurin to resorufin is proportional to aerobic respiration.
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. Resazurin is dark blue in color and has little intrinsic. In alamarblue assay the growing cells cause a chemical reduction of the alamarblue dye from nonfluorescent blue to red fluorescent. A 96well plate containing the cells and the compounds to be tested is prepared using standard methods. Microplate alamar blue assay for susceptibility testing of candida albicans. These findings suggest that the alamar blue assay may not be appropriate for studying proliferation in tendonderived cells. Alamarblue contains resazurin and additional compounds to prevent the overreduction of resazurin to a nonfluorescent product.
Investigation of the alamar blue resazurin fluorescent. A simple method to measure cell viability in proliferation and cytotoxicity assays abstract. Pdf the use of alamar blue assay for quantitative analysis of. The bioassay may also be used to establish relative cytotoxicity of agents within various chemical classes 3. This results in colorimetric absorbance and fluorescence changes. Pdf application and evaluation of the alamarblue assay for. Pdf cell viability assessment using the alamar blue assay.
Briefly, 2 groups of bmscs at passage 2 were seeded in 24well plates at a density of 5. Pettit, microplate alamar blue assay for susceptibility testing of candida albicans biofilms, medical mycology, volume 45, issue 7. Upon entering living cells, resazurin is reduced to resorufin, a compound that is red in color and highly fluorescent. Optimized alamarblue assay protocol for drug doseresponse. This assay has excellent performance compared to other resazurinbased cell proliferation kits such as alamarblue, prestoblue, or celltiterblue. Similar to alamarblue reagent, alamarblue hs reagent displays an extended viability detection time window for a wide variety of organisms and thus can be used with various human and animal cell lines, bacteria, plant, and fungi, resulting in quantitative measurement of cell viability and proliferation. The number of cells per well were 40, 80, 400, 800, 4000 and 8000. This application note demonstrates how to measure an alamarblue assay for assessment of cell proliferation using a bmg labtech plate reader. Alamarblue cell viability reagent from thermo fisher scientific.
Fluorescence measurements are more sensitive than absorbance measurements and hence are the. Suitable for colorimetric or fluorometric detection. Cell proliferation was assessed by comparing the results of an alamar blue assay and two dna quantification assays, cyquant and picogreen, on two human cancer cell lines and two human primary cell sources. The alamar blue assay reagent which also contains resazurin was added to fresh media with and without the doi and incubated for 20 min. Resazurin dye has been broadly used as indicator of cell vi ability in several types. Alamarblue assay for cell proliferation bmg labtech. Then the plates are read with a spectramax gemini xs microplate fluorometer molecular devices cooperation, sunnyvale, ca, usa using an excitation wave. The blue nonfluorescent resazurin reagent is reduced to highly fluorescent resorufin by dehydrogenase enzymes in metabolically active cells. Alamar blue, choriocarcinoma, invasion, migration, viability. The resazurin cell viability kit is a fluorescent assay that detects cellular metabolic activity. Here, we optimized the alamarblue assay standard protocol to result in a more precise and reliable assay for drug efficacy testing in spheroid cultures using an optimized fluorescencebased metabolic assay. A simple method to measure cell viability in proliferation.
Analysis of cell viability by the alamarblue assay request pdf. Accumulation of reduced alamar blue into cell culture media hepg2 cells were seeded at several densities from 40 to. Pdf application and evaluation of the alamarblue assay. Store at 20c resazurin cell viability kit description. The absorbance spectrum for reduced and oxidized forms of the resazurin dye are highlighted in figure 1.
The alamarblue dye is a redox indicator that yields a colorimetric change and a fluorescent signal in response to metabolic activity. The celltiterblue cell viability assay provides a homogeneous, fluorometric method for estimating the number of viable cells present in multiwell plates. This is a trusted and established reagent which has been available since 1993. Living cells are metabolically active and are able to reduce via mitochondrial reductase, the nonfluorescent dye resazurin to the stronglyfluorescent dye resorufin fig. Cell proliferation assays are essential to developing an understanding of the molecular mechanisms that modulate cell growth and differentiation. Alamar blue ab is a watersoluble dye that has been previously used for quantifying in vitro viability of various cells fields and lancaster, 1993. Using the well established alamar blue assay, the study demonstrates how the transfer from 2d substrates to 3d collagen matrices can affect the uptake of the resazurin itself, affecting the outcome of the assay. Fluorescence left and absorbance right spectra of thermo scientific alamarblue reagent in oxidized and reduced states. Overview alamarblue is a cell viability assay reagent which contains the cell permeable, nontoxic, and weakly fluorescent blue indicator dye called resazurin. The cell proliferation kit i mtt can be used for multiple applications, such as, quantification of cell growth and viability. The microplate alamarblue assay maba is a sensitive, rapid, inexpensive, and nonradiometric method, which evaluates metabolic function and cellular health and offers the potential for screening large numbers of antimicrobial compounds. A simple method to measure cell viability in proliferation scielo.
Since the autoclaving procedure was not efficient in reducing the resazurin. Basal medium without cells and serum medium without cells. Samples of alamar blue were read at the lambda max lambda max was the same for resazurin and ab and the concentration of alamar blue calculated from the standard curve of resazurin. Absorbance values may be affected by the type of plate whether round or flat bottom and the plate manufacturer. Inhibition of osteogenic and adipogenic potential in bone. Cytokine assays measure cytokineinduced proliferation, recover and expand cells at the end of the study if desired alamarblue reagent as a cell viability assay reagent the alamarblue assay is based on the ability of metabolically active cells to convert the reagent into a fluorescent and colorimetric indicator.
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