ROS Detection in Botryococcus braunii Colonies with CellROX Green Reagent

Background

Among the first methods developed to detect and quantify hydrogen peroxide and other organic hydroperoxides was the use of titanium (IV) ion (MacNevin and Urone, 1953). The yellow color resulted from the completion of titanium (IV) and peroxide molecules was detected by colorimetry. This method was used to detect endogenous peroxides, and to assay the catalase activity in two varieties of pear fruits for correlation with fruit ripening (Brennan and Frenkel, 1977). Another method to detect lipid hydroperoxides is based on thiobarbituric acid (TBA) and was used to measure the deterioration of foods such as milk (Sidwell et al., 1955). Although this method did not use organic solvents, steam distillation of an acidified slurry was necessary to detect hydroperoxides, and the resulting red color was quantified spectrophotometrically. The procedures described above have several disadvantages such as low sensitivity, interference with other compounds, and use of solvents or substances which may damage the living cells. A more sensitive method was developed in which the blue fluorescence of scopoletin (6-methyl-7-hydroxy-1:2-benzopyrone) disappeared after its oxidation by peroxidase enzyme (Andreae, 1955; Perschke and Broda, 1961). This method was used to detect the H₂O₂ production by NADPH in the microsomes from rat liver (Thurman et al., 1972). However, scopoletin is expensive, difficult to extract, and is an extremely toxic natural compound (Ojewole and Adesina, 1983a and 1983b). On the other hand, fluorescein is a dye chemically synthesized (Baeyer, 1871) and the chemical structure was elucidated (Markuszewski and Diehl, 1980). The fluorescence of both compounds, scopoletin and fluorescein, was then explained based on their similar chemical structure. So, further development of novel fluorescent dyes more stable and versatile allowed their use in very specific applications (Cathcart et al., 1983). For instance, 2,7-dichlorohydro-fluorescein diacetate (DCFH-DA) was used to study the intracellular production of active oxygen in the brown alga Fucus evanescens (Collén and Davison, 1997). The same compound DCFH-DA was also used to detect oxidative stress tolerance by abscisic acid (ABA) in the green microalga Chlamydomonas reinhardtii (Yoshida et al., 2003). Due to the wide application of these fluorescent dyes, private companies developed other compounds with different properties and each designed for specific applications. CellROX Green Reagent was designed to detect the production of ROS in living cells. So, we chose this dye to detect ROS in early times in B. braunii living cells (Life Technologies Corp., 2012). These reagents are cell-permeable and show no or very weak fluorescence in a reduced state, but their oxidation results in a strong fluorescence. In presence of ROS, the CellROX Green Reagent undergoes oxidation and produce green fluorescence followed by its binding to the DNA in the nucleus. This fact allows us to distinguish between the fluorescence resulting from ROS and the fluorescence from the chlorophyll molecule. Furthermore, this reagent can be fixed with formaldehyde and is compatible with some detergents. These characteristics of CellROX Green Reagent made it suitable to analyze ROS production in stress conditions in cells of the colonial microalga Botryococcus braunii race B (Nonomura, 1988; Banerjee et al., 2002).