Alamar blue

Alamar Blue (AB) Live cells resazurin reduction to red fluorescent dye resorufin Cell metabolism No [33, 34]... 

However, fullerene C60-modified surface is adequate for the adhesion and normal growth of cells in culture. Cells of the line MA-104 in the Eagle-MEM medium formed on fullerene film a normal monolayer. Cellular viability was assessed with the resazurin (Alamar Blue) reduction test. The dye resazurin is reduced by mitochondrial dehydrogenases of viable cells into the fluorescent product resorufin (maximum X = 530 nm, max X = 590 nm). The intensity of... 

Cells were grown for 24 h, growth medium was replaced for serum-free medium and the appropriate preparations were introduced, plates were illuminated with halogen lamp (about 45mW/cm2) and further incubated for 18 h in the darkness. Viability was assessed by Alamar Blue reduction. Results are presented as percent of control. 

Andrews MJ, Garle MJ, Clothier RH et al. (1997) Reduction of the new tetrazolium dye, Alamar Blue , in cultured rat hepatocytes and liver fractions. ATLA. 25 641-653. 

Clothier R, Starzec G, Pradel L et al. (2002) The prediction of human skin responses by using the combined in vitro fluorescein leakage/Alamar Blue (Resazurin) assay. ATLA. 30 493-504. 

Before further testing and to confirm that the compounds are cytotoxic rather than merely interfering with the Alamar blue indicator dye, they are re-bioassayed using two other indicator dyes. Calcein-AM is a fluorescent dye that measures changes in cell mem brane permeability, an indicator for one of the penultimate steps of cell death, uci e e measures the amount of adenosine triphosphate (ATP) synthesis in a chemilurmne assay. For some compounds, cell death was also confirmed by microscopic exami Papanicolaou-stained cell preparations.11... 

Shown in Figure 6 is in vitro cytotoxic activity of PIPAAm-PBMA micelles loaded with ADR or micelles without ADR at 29°C (below the LCST) and at 37°C (above the LCST) compared with that of free ADR. In vitro cytotoxic activity was measured using bovine aorta endothelial cells. Bovine aortic endothelial cells were obtained as previously reported using dispase for cell dissociation from freshly harvested bovine aorta [13]. The cells plated at a density of 3 x cells/well, were exposed with free ADR or micelles loaded with ADR at below and above the LCST for 5 days. In order to assay cytotoxicity of the free ADR or micelles loaded with ADR, culture medium was replaced with 10% FBS-supplemented phenol red-free DMEM containing 10% alamar Blue, a dye that is subject to reduction by cytochrome c activity and changes the color from blue to red [38]. After 4-hour incubation, reduction of the dye was estimated by absorbance at 560 and 600 nm. PIPAAm-PBMA polymeric micelles loaded with ADR showed higher cytotoxic activity than that of free ADR at 37°C (above the LCST)... 

Failure Marked impairment of energy homeostasis with volume contraction impaired mitochondrial reductive activity with decreased ATP concentration (e.g., MTT, Alamar blue) organelle and cell swelling and distortion, cell lysis with intracellular enzyme release (e.g., LDFf release)... 

Dye oxidation (e.g., tetrazolium reductase activity with 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide, MTT 2-[4-iodophenyl]-3-[4-nitrophenyl]-5-[2,4-disulfophenyl]-2H tetrazolium monosodium salt, WST-1 3- (4,5 -carboxymethoxyphenyl) -2-(4-sulfophenyl)-2 H-tetra-zolium, MTS 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide inner salt, XTT 2,2 -di-p-nitrophenyl-5,5 -diphenyl-3,3 -(3,3 -dimethoxy-4,4 -diphe-nylenej-ditetrazolium chloride, NET), Alamar blue assays, ATP concentration (e.g., luciferase assay), oxygen consumption (e.g., oxygen electrodes, phosphorescent oxygen-sensitive dyes), mitochondrial protein and nucleic acid synthesis mitochondrial mass (e.g., mitotracker dyes) mitochondrial membrane potential (e.g., tetramethylrho-damine methyl ester, TMRM tetramethylrhodamine ethyl ester, TMRE)... 

Alamar blue assay, Tetrazole reduction (MTT, WST assay) Tritium-thymidine (3H-TdR) incorporation, bromodeoxyuridine (BrdU) incorporation... 

There are many ways to estimate microbial growth. The simplest is visual inspection of colonies growing on agar plates, though this method is difficult to adapt for HTS. There is a well-known correlation between cell density and optical density, which can be exploited in a 96-well microtiter plate format (e.g., [43]). Measurement of the incorporation of radioactive nutrients is an excellent quantitative method, but has fallen from favor due to concerns about spills and contamination. Finally, both spectrophotometric and fluorimetric assays are conveniently adapted to HTS formats, and Alamar blue is only one example of the tools available for this purpose. As mentioned, we have even found it convenient to use simple visual inspection of Alamar blue plates to identify wells of interest. However, quantification obtainable with a microplate reader is attractive in many settings. 

LA Collins, SG Franzblau. Microplate Alamar blue assay versus BACTEC 460 system for high-throughput screening of compounds against Mycobacterium tuberculosis and Mycobacterium avium. Antimicrob Agents Chemother 41 1004-1009,1997. 

Kinase-dependent cell growth BaF3 Cell number or metabolism measured by standard cell detection methods such as ATP detection or MTT dye, Alamar blue cell growth dependent upon specific tyrosine kinase Very high-throughput cell-based assay Limited for use with tyrosine kinases cell line must be made Warmuth (2007)... 

Proliferation of the BaF3 cells can be monitored rapidly by using standard techniques such as with Alamar blue or the commercially available CellTiter-Glo. These systems measure cell numbers by detecting intracellular ATP concentrations or by measuring intracellular reduction of the Alamar blue dye. The BaF3 system is excellent for looking at larger kinase constructions and receptor tyrosine kinases (Warmuth et al., 2007). 

FIGURE 6.4 Comparison of effects of Alamar blue (resazurin) and GF-AFC reagents on viability of cells measured using a luminescent ATP assay. Resazurin or GF-AFC was incubated with 10,000 DU145 cells per well for 18 hr prior to measuring ATP as an indicator of cell viability. Alamar blue reagent is more toxic to cells. 

Mitochondrial (MTT, XTT, Alamar blue, ATP) ribosomal ( C-Leu or -URI incorporation) lysosomal (neutral red uptake/release) nuclear H-Thy incorporation, DNA binding)... 

O Brien, P.J., I. Wilson, T. Orton and F. Pognan. Investigation of the alamar Blue (resazurin) fluorescent dye for the assessment of mammalian cell cytotoxicity. Eur. J. Biochem. 267 5421-5426, 2000. 

Schirmer K, Herbrick JS, Greenberg BM, et al. 1997. An evaluation of the cytotoxicity and photocytotoxicity of intact and photomodified creosote through the use of a rainbow trout gill cell line, TRGILL-W1, and two flourescent indicator dyes, alamar blue and 5-carboxyfluorescein diacetate acetoxymethyleste. Can Tech Rep Fish Aquat Sci 2192 4. 

Two assays were developed that measure the potency of the FGF-4 transgene carried by Ads FGF-4. In the first case, a one-step growth-promotion assay is conducted on normal, human retinal pigment epithelial cells (ARPE-19). The assay measures metabolic activity (Alamar blue dye metabolism) following infection of ARPE-19 cells with a serial dilution of the virus. The increase in metabohc achvity was measured in relation to a mock-infected control. This increase correlates with FGF-4 produchon determined by an FGF-4 ELISA, increased de-novo DNA synthesis measured by BrdU incorporation, and an increase in cell number. This procedure is therefore an appropriate in-vitro efficacy measure, indicating that the FGF-4 transgene product is biologically active. 

Alamar blue dye metabolism 168 Albuterol 59 Aldurazyme 24, 475 Alefacept 455... 

Hamid, R., Rotshteyn, Y., Rabadi, L., Parikh, R., and Bullock, P. (2004) Comparison of alamar blue and MTT assays for high through-put screening. Toxicol, in Vitro,... 

Compound Bioautography Agar Well Diffusion Assay Serial Broth Dilution Assay Alamar Blue Assay ... 

In vitro quantitative antibacterial activity was evaluated using Alamar Blue Assay [56]. It is a colorimetric oxidation-reduction assay which involves the addition of Alamar blue dye as an indicator. It evaluates the metabolic activity of the microorganisms. The activity of the evaluated drug is expressed as minimum inhibitory concentration ( 0,g/ml). Sampangine, SAMM2 and SAM MMl were tested for their activity against Staphylococcus aureus ATCC 6538, Pseudomonas aeruginosa ATCC 15442, Bacillus subtilus ATCC 6633, and E. coli ATCC 10536. Streptomycin was used as a positive control. 

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