Resazurin assay

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. 

Both the neutral red and SRB staining methods present the advantages that they avoid some potential for artifacts related to chemical interference with the tetrazolium and resazurin assay chemistries used to measure cell metabolism and the signals are stable. The obvious and major disadvantage of... 

The recent identification of selective protease substrates to simultaneously measure markers of both viable and dead cells led to the development of optional methods for HTS that provide flexibility and added advantages (Niles et al. 2007a). The assay to measure viable cells is based on a cell-permeable protease substrate called glycyl-phenylalanyl-aminofluorocoumarin (GF-AFC). The procedure is a homogeneous add-incubate-measure method that is faster, more sensitive, and less toxic to cells than the tetrazolium and resazurin reduction assays. The substrate can be prepared in an aqueous buffer and is added directly to samples containing cells. The substrate permeates viable cells where constitutive protease activity in the cytoplasm rapidly removes the amino acids, yielding free AFC. The amount of AFC released is directly proportional to viable cell numbers and shows improved sensitivity compared to the resazurin assay (Figure 6.5). The AFC is detected via a microplate fluo-rometer equipped with a (380- to 400-nm excitation/505-nm emission) filter set. 

Fluorescence assays such as calcein staining provide information about viability of cells. The resazurin assay is based on the metabolism of this compound to render a fluorescent dye which is retrieved from the cell culture with the perfundate and may be analyzed to determine the niunber of viable cells present in the culture. 

The cytotoxicity of these compounds was studied on HeLa cells in a resazurin assay. Bisphosphonate 62 and the diastereomeric mixture of bisphosphonothioate 63 exhibit extraordinarily low cytotoxicity. They do not show cytotoxic effects up to a concentration of 20 mM (Figure 2.7) and are thus far less toxic than the boron compounds which are currently employed in... 

Moreover, following the Resazurin assay, healthy growth of L929 mammalian cells was observed. The ability of the mammahan cells to adhere to blended films points to desirable biocompatibility with the new functional films (Figure 4.8). 

Duarte M et al (2009) A quantitative resazurin assay to determinate the viability of Trichomonas vaginalis and the cytotoxicity of organic solvents and surfactant agents. Exp Parasitol 123 195-198... 

DeFelip and coworkers use sodium resazurin as a dye to give a rapid assay in three to six hours 33. 

Resazurin is a chemical redox indicator that functions like the tetrazolium compounds. The resazurin reduction assay approach has successfully replaced tritiated thymidine incorporation for some HTS applications (Ahmed, Gogal, and Walsh 1994 Shum et al. 2008). Resazurin can be dissolved in physiological buffers resulting in an intense blue solution that is added directly to growing... 

One example is the known interference by reducing compounds that affect the chemical conversion of substrate to a colored indicator. This is especially true for the tetrazolium assays (Ulukaya, Colakogullari, and Wood 2004 Chakrabarti et al. 2000 Pagliacci et al. 1993 Collier and Pritsos 2003). The growing list of interfering compounds includes ascorbic acid and sulfhydryl reagents such as glutathione, coenzyme A, dithiothreitol, etc. Similar interferences by compounds that affect the oxidation and reduction chemistry of cells are likely to cause artifacts with the resazurin reduction assay. Assays that measure markers of metabolism also can be influenced by the pH of the culture medium and other factors that may stimulate or stress the metabolic rates of cells. 

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. 

FIGURE 6.5 Comparison of sensitivity of resazurin reduction and GF-AFC cleavage assays for detection of numbers of cells. Values are plotted as signal-to-noise (S N) ratios. Comparison of data at S N = 3 indicates approximately 30-fold better sensitivity of the GF-AFC assay after 30 min of incubation compared to resazurin incubated for 3.5 hr. 

Frequently, the first sign of hepatotoxicity is breakdown of the outer cell plasma membrane. If the cell membrane is full of holes , it allows the internal contents of the hepatocyte to leak out into the culture medium. By taking an ahquot of the cell culture supernatant following exposure to a toxic chemical and incubating the sample with a reagent that recognizes LDH, the amount of LDH that leaks out of the damaged cell can be determined. This assay is based on a coupled enzymatic assay that results in the conversion of resazurin into the fluorescent compound, resorufm. 

Nateche, F., Martin, A., Baraka, S., Palomino, J.C., Khaled, S. and Portaels, F. (2006). Application of the resazurin microtitre assay for detection of multidrug resistance in Mycobacterium tuberculosis in Algiers. Medical Microbiology, 55 857-860. 

The calibration curve was obtained using optimized concentrations of formaldehyde dehydrogenase, NAD, diaphorase, resazurin and buffer pH. The calibration curve measured at 1 minute after injection fits the equation Y 120 (X) + 4.68 as shown in Figure 4. This figure also shows the extended calibration plot at low concentrations and the data fit the equation Y 0.437 (X) + 11.3. The lowest concentration of formaldehyde in an assay... 

More recently, the sensitivity of enzymatic assays has been further increased by a number of coupling methods (Table 2). These transfer hydrogen from NADH to acceptor compounds, such as a tetrazolium dye to produce diformazan (Mil) or the dye resazurin to produce resorfin (M9). The enzyme... 

Altered cell permeability assays The loss of cell membrane integrity can result in the release of certain soluble, cytosolic enzymes Lactate dehydrogenase release assay (tetrazolium salt-based assay) Glucose-6-phosphate dehydrogenase release assay (resazurin-based assay)... 

Zhang, H. Du, G. Zhang, J. Assay of mitochondrial functions by resazurin in vitro. Acta Pharmacol. Sinica 2004, 25, 385-389. 

Zrimsek, R Kune, J. Kosec, M. Mrkun, J. Spectrophotometric application of resazurin reduction assay to evaluate boar semen quality. Int. J. Androl. 2004,27, 57-62. 

Recently, a universal enzyme-coupled fluorescence assay for glycosyl transferases was developed. This method is extremely cost-effective and is based on the quantification of nucleotides produced in the glycosyl transfer reaction. The guanosine diphosphate (GDP), uridine diphosphate (UDP), and cytidine monophosphate (CMP) are phos-phorylated with nucleotide kinase in the presence of excess of ATP, generating ADP. Via coupled enzyme reactions involving ADP-hexokinase,glucose-6-phosphate dehydrogenase, and diaphorase, the ADP is utilized for the conversion of resazurin to resorufin, which is then quantified by fluorescence measurement. 

Assay Medium. Herrell el al. (16) recommend the use of fluid thioglycol-ate broth with the following composition yeast extract 5 g., Bacto-casitone 15 g., glucose 5 g., sodium chloride 2.5 g., Z-cystine 0.75 g., thioglycollic acid 0.3 ml., agar 0.75 g., resazurin (certified) 0.001 g., and distilled water 1 liter the final pH of this medium should be 7.1. 

The sensitivity was increasec s geral times by the use of fluorimetric determination of NADH. However the preliminary extrac on of BA from serum was still required. In 1976 Mashige et al. reported the first direct enzymatic-fluorimetric assay, based upon the use of the 3 ci -HSD in combination with a resazuri-ne-diaphorase system. The hydrogen of the generated NADH is transferred by diaphorase to resazurine, to yeld the fluorophore, resorfin. The fluorescence of resorfin is then measured at 580 nm with excitation at 560 nm. This method introduced a significant improvement in sensitivity (0.5 mol/l) and clinical usefulness, since no preliminary extraction was required. 

---