Resazurin reduction assay

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. 

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. 

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. 

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. 

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