The correlates and alleged biochemical background of the resazurin reduction test in semen

Abstract

Reduction of the blue dye resazurin to pink resorufin is used to estimate the concentration of metabolically active spermatozoa in semen samples. In order to quantify the reduction of resazurin, a spectrophotometric method was developed measuring the change from blue to pink in the butanol extracted colour. The biochemical mechanisms involved in the reduction of resazurin by motile spermatozoa and seminal plasma were investigated. Addition of NADH + H+ to sperm suspension or seminal plasma increased the reduction of resazurin. The reduction reaction was inhibited by high concentrations of dicoumarol, a specific inhibitor of the diaphorase enzyme, in a dose-dependent manner. It is suggested that the sperm diaphorase enzyme transfers electrons from NADH + H+ to resazurin, reducing it to resorufin. The degree of resazurin reduction was strongly correlated with the concentration of motile spermatozoa recovered from the 90% Percoll fraction (r = 0.98, p < 0.001). A positive correlation was also found between the reducing capacity of seminal plasma (n = 62) on the one hand, and sperm concentration (r = 0.72, p < 0.0001), progressive motility (r = 0.45, p < 0.01), normal morphology (r = 0.50, p < 0.01), and gamma-glutamyltransferase (r = 0.36, p < 0.05) on the other hand. These findings, together with our previous observations that the reduction reaction is inhibited by reactive oxygen species and polymorphonuclear white blood cells, increase our understanding of the biochemical basis of the resazurin test and may provide better insight into the interpretation of this test.

A comparative study of colorimetric cell proliferation assays in immune cells

“…For instance, the cytosolic NADPH-oxidoreductases catalyze the conversion of MTT to insoluble formazan with the color of visible Herein, detergents such as SDS and NP-40 led to different results; thus, the careful setting of procedures is required. In addition, resazurin may be reduced by ''diaphorase'' enzymes, including dihydrolipoamide dehydrogenase, quinine oxidoreductase, and flavin reductase (Zalata et al 1998). Differences in reducing enzymes could lead to different outcomes in the cell viability test for anti-cancer chemical compounds (Hamid et al 2004).…”

Essential design considerations for the resazurin reduction assay to noninvasively quantify cell expansion within perfused extracellular matrix scaffolds

“…In contrast, for MDCK-recellularized kidney scaffolds, the FI peaked and then gradually decreased for the remainder of the assay, decaying to zero by 24 hours (Figure 2A inset). The decrease in perfusate FI may be attributed to further reduction of resorufin to hydroresorufin, a non-fluorescent, colorless compound (Figure 1) [26, 27]. Between 3 and 9 hours, there was an obvious color change in the MDCK cell perfusate from pink to pale orange, and this occurred regardless of whether working solutions contained AlamarBlue or resazurin alone (Figure 2B).…”

“…The irreversible reduction of resazurin to resorufin is mediated by intracellular diaphorase enzymes [26, 27] and generates a strong fluorescent signal that may be measured using a spectrophotometer to noninvasively provide a comprehensive assessment of cellular metabolic activity within a population of cells. The resazurin reduction assay is inexpensive and non-toxic to cells at low concentrations and brief incubation periods (i.e., <4 hours [28]), and is therefore a useful method to indirectly measure cell proliferation kinetics.…”

“…This is particularly important for applications involving engineered tissues or biomaterial scaffolds with high cell densities due to the layered geometry of 3D cell culture. Moreover, while the primary reduction of resazurin to resorufin is irreversible, an important, but lesser-known, byproduct may be generated from a secondary reaction that depletes resorufin and produces the colorless and non-fluorescent hydroresorufin (Figure 1) [26, 27]. These factors may cause the resazurin assay to greatly underestimate the actual number of cells present in cell-dense biomaterial scaffolds.…”

In Vitro Cell Death Discrimination and Screening Method by Simple and Cost-Effective Viability Analysis

“…resazurin, AlamarBlue ® , CellTiter-Blue ® [7, 11, 20, 21]), enzyme activity (conversion of tetrazolium salts such as MTT, MTS, WST [22, 23]) and adenosine triphosphate (ATP) levels [11, 24, 25]. The resazurin assay is based on the reduction of the oxidised, non-fluorescent, blue state (resazurin) to the fluorescent, pink state (resorufin) by components of the mitochondrial electron transport chain and mitochondrial and cytoplasmatic enzymes of viable and metabolically active cells (see [26-28] for details). A decreased rate of reduction (resazurin conversion) therefore indicates impairment of cellular metabolism [27].…”