Application and evaluation of the alamarblue assay for cell growth and survival of fibroblasts

Voytik‐Harbin, Sherry L.; Brightman, Andrew O.; Waisner, Beverly; Lamar, Carlton H.; Badylak, Stephen F.

doi:10.1007/s11626-998-0130-x

Cited by 140 publications

(104 citation statements)

References 23 publications

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“…The commercialized Alamar blue dye contains 0.1 mg/ml of resazurin. The manufacturer's report and most other studies suggested the use of one-tenth dilution of Alamar blue so, for our study, we opted for a final concentration of 0.01 mg/ml of resazurin (5,11,16,32).…”

Section: Discussionmentioning

confidence: 99%

“…It has been commercialized since 1993 as Alamar blue dye (10) as a viability cell test and has been assessed several times on different types of cells for its cytotoxicity reliability, i.e. fibroblasts (11), immortalized and cancer cell lines (5,12), tumor necrosis factor-hypersensitive cells (13), and for its cell proliferation reliability on mouse and human lymphocytes (6,14) and primary neuronal cell culture (15). Viability measurement using resazurin is similar to or greater than traditional tetrazolium salts (MTT, XTT) and [ 3 H] thymidine assay techniques (5,10,11,16).…”

mentioning

confidence: 99%

“…fibroblasts (11), immortalized and cancer cell lines (5,12), tumor necrosis factor-hypersensitive cells (13), and for its cell proliferation reliability on mouse and human lymphocytes (6,14) and primary neuronal cell culture (15). Viability measurement using resazurin is similar to or greater than traditional tetrazolium salts (MTT, XTT) and [ 3 H] thymidine assay techniques (5,10,11,16). Recently, it has been shown that resazurin metabolism assay gives better results in the evaluation of lens viability than the 5-carboxy-fluorescein diacetate acetomethyl ester which is an esterase substrate that is converted by the non-specific esterases of living cells into carboxy fluorescein (17).…”

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confidence: 99%

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A new nondestructive cytometric assay based on resazurin metabolism and an organ culture model for the assessment of corneal viability

Perrot¹,

Dutertre-Catella

Martin³

et al. 2003

Cytometry Pt A

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Background: Three-dimensional culture or human corneal equivalents for safety testing are difficult to investigate with classic cytometric or biochemical methods. So a fluorometric method is proposed using resazurin probe. Methods: Absorbance and fluorescence spectra of the oxidized and reduced forms of resazurin were performed to determine optimal measurement conditions. More than 100 enucleated porcine eyes were used for this experiment. Twelve corneas were immersed in resazurin solution and fluorescence was measured hourly from 1 to 10 h. Ninety benzalkonium chloride-treated corneas and control corneas were used as toxicity controls, and corneal viability was compared with in vivo rabbit eye irritation. Results: After analysis of spectra, the optimal measurement condition of resazurin metabolism proved to be a fluorescence measurement using 570 nm excitation wavelength and 590 nm emission wavelength. The reduction of

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

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confidence: 99%

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A new nondestructive cytometric assay based on resazurin metabolism and an organ culture model for the assessment of corneal viability

Perrot¹,

Dutertre-Catella

Martin³

et al. 2003

Cytometry Pt A

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“…The droplets' chemical isolation was used to implement a single-cell performancebased assay. For this, we chose to investigate the metabolic activity of fibroblasts (NHDF) using the alamarBlue assay, which is used routinely to estimate the number of metabolically active cells in bulk (47). In the presence of a live cell, its fluorescence turnover linearly increases over time.…”

Section: E)mentioning

confidence: 99%

Stationary nanoliter droplet array with a substrate of choice for single adherent/nonadherent cell incubation and analysis

Shemesh

Ben-Arye

Avesar³

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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Microfluidic water-in-oil droplets that serve as separate, chemically isolated compartments can be applied for single-cell analysis; however, to investigate encapsulated cells effectively over prolonged time periods, an array of droplets must remain stationary on a versatile substrate for optimal cell compatibility. We present here a platform of unique geometry and substrate versatility that generates a stationary nanodroplet array by using wells branching off a main microfluidic channel. These droplets are confined by multiple sides of a nanowell and are in direct contact with a biocompatible substrate of choice. The device is operated by a unique and reversed loading procedure that eliminates the need for fine pressure control or external tubing. Fluorocarbon oil isolates the droplets and provides soluble oxygen for the cells. By using this approach, the metabolic activity of single adherent cells was monitored continuously over time, and the concentration of viable pathogens in blood-derived samples was determined directly by measuring the number of colony-formed droplets. The method is simple to operate, requires a few microliters of reagent volume, is portable, is reusable, and allows for cell retrieval. This technology may be particularly useful for multiplexed assays for which prolonged and simultaneous visual inspection of many isolated single adherent or nonadherent cells is required.single cell | nanoliter array | diagnostics C ommon single-cell analysis methods, such as flow cytometry and mass cytometry (1), offer high throughput and accurate single-cell marker quantification, yet they lack the ability to monitor large numbers of single cells continuously and simultaneously in performance-based assays (2, 3). Conventional microscopy may be used for these assays; however, in the case of single cells, they cannot analyze extracellular events, such as secretion. To achieve this, cells must be isolated in compartments that can sustain cell viability and growth while permitting conventional optical analysis over many hours to days. Dropletbased microfluidics, which enables single-cell encapsulation in nano-and subnanoliter droplets by surrounding microscopic aqueous medium with an immiscible carrier fluid (4-8), recently gained interest with the appearance of digital PCR (9-11). Much of the work thus far has been directed toward improving droplet manipulation capabilities (12-16). With these methods, droplets are mobile, and thus cytometry is performed under flow conditions (17), making continuous monitoring of single cells difficult. Continuous monitoring may be achieved by using stationary indexed droplets, but many current droplet immobilization techniques are limited by pressure coupling between droplet generation and capture events, as well as the requirement to adjust droplet volume to nanowell size (6,18,19). The vast majority of methods used to generate water-in-oil droplets begin by priming a continuous oil phase in a microfluidic channel followed by an injection of a dispersed (aqueous) medium (2...

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“…Cells were treated with 10 nmol/L Δ4A or 1 nmol/L E 2 and various concentrations of test compounds (0.2, 1, 3, 10, and 50 μmol/L) for 3 d or 6 d. Each concentration was tested in triplicate. The proliferation assay was performed using the alamarBlue® assay as previously described [33,34] . A Safire 2 microplate reader (Tecan, Switzerland) was used to detect the fluorescent intensity at Ex 560 nm/Em 590 nm.…”

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confidence: 99%

Discovery of novel aromatase inhibitors using a homogeneous time-resolved fluorescence assay

Lao

et al. 2014

Acta Pharmacol Sin

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Aim: aromatase is an important target for drugs to treat hormone-dependent diseases, including breast cancer. The aim of this study was to develop a homogeneous time-resolved fluorescence (HTRF) aromatase assay suitable for high-throughput screening (HTS). Methods: A 384-well aromatase HTRF assay was established, and used to screen about 7000 compounds from a compound library. Anti-proliferation activity of the hit was evaluated using alamarBlue(R) assay in a hormone-dependent breast cancer cell line T47D. Molecular docking was conducted to elucidate the binding mode of the hit using the Discovery Studio program. Results: The Z′ value and signal to background (S/B) ratio were 0.74 and 5.4, respectively. Among the 7000 compounds, 4 hits (XHN22, XHN26, XHN27 and triptoquinone A) were found to inhibit aromatase with IC 50 values of 1.60±0.07, 2.76±0.24, 0.81±0.08 and 45.8±11.3 μmol /L, respectively. The hits XHN22, XHN26 and XHN27 shared the same chemical scaffold of 4-imidazolyl quinoline. Moreover, the most potent hit XHN27 at 10 and 50 μmol/L inhibited the proliferation of T47D cells by 45.3% and 35.2%, respectively. The docking study revealed that XHN27 docked within the active site of aromatase and might form a hydrogen bond and had a π-cation interaction with amino acid residues of the protein.Conclusion: XHN27, an imidazolyl quinoline derivative of flavonoid, is a potent aromatase inhibitor with anti-proliferation activity against breast cancer in vitro. The established assay can be used in HTS for discovering novel aromatase inhibitor.Keywords: aromatase; breast cancer; imidazolyl quinoline; triptoquinone A; homogeneous time-resolved fluorescence assay; highthroughput screening; molecular docking; drug discovery Acta Pharmacologica Sinica (2014Sinica ( ) 35: 1082Sinica ( -1092 doi: 10.1038/aps.2014 [12,13] . Previous studies have demonstrated that AIs provide an increased survival benefit compared with other therapies and have acceptable toxicity profiles with decreased virginal bleeding and thromboembolism and increased rash, diarrhea and vomiting [14,15] . As AIs sometimes have more severe bone, brain and heart side effects, research for alternative compounds is necessary [15][16][17] .Natural products, extracted from traditional medicines and foods, may be helpful for discovering novel AIs that may selectively target aromatase in the breast and reduce systemic toxicity [18] . Among these compounds, flavonoids [19] are the most commonly investigated agents due to their prominent aromatase inhibitory activity and high breast selectivity [18] . Moreover, flavonoids may modulate the multi-step process of carcinogenesis through cellular and molecular mechanisms [19] . Biochanin A (BCA), isolated from red clover (Trifolium pretense), is a common isoflavone product that can inhibit aromatase activity and cell growth in MCF-7 cells [20] . Furthermore, cyp19a1 mRNA abundance was significantly reduced by BCA through promoter regulation in SK-BR-3 cells [20] .The classical tritiated water release assay [21,22] is ...

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Application and evaluation of the alamarblue assay for cell growth and survival of fibroblasts

Cited by 140 publications

References 23 publications

A new nondestructive cytometric assay based on resazurin metabolism and an organ culture model for the assessment of corneal viability

A new nondestructive cytometric assay based on resazurin metabolism and an organ culture model for the assessment of corneal viability

Stationary nanoliter droplet array with a substrate of choice for single adherent/nonadherent cell incubation and analysis

Discovery of novel aromatase inhibitors using a homogeneous time-resolved fluorescence assay

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