A new type of O2−-generating tool for oxidative stress studies by remodeling neutrophil NADPH oxidase

Induction of apoptosis in Caco-2 cells by exogenously added O2− produced by a nanodevice

“…When the cells were cultured for 24 h in the presence of Device II and NADPH, the percentage of surviving cells estimated by the Trypan blue exclusion test was 19% relative to the control cells (Fig. 1 generating Device I [9]. To confirm the results described above, MTT assay was also performed with the cells treated with or without Device II (Fig.…”

“…Briefly, Device I [9] was diluted 10-fold with 50 mM PIPES (pH 7.0) containing 8 mM MgCl 2 and 10 μM FAD, and then 10 mM 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and 5 mM N-hydroxysulfosuccinimide were added slowly in that order with gentle stirring and allowed to stand at 25 1C for 30 min (with stirring every 10 min). After the reaction, the mixture was dialyzed against 50 mM PIPES buffer (pH 7.0) containing 10 μM FAD and 20% glycerol at 4 1C for 4 h. The resulting mixture was designated as Device II and frozen in aliquots until use.…”

“…To examine how O 2 À affects cells, a tool that can constantly produce O 2 À at a high rate is required. Xanthine oxidase has sometimes been used for this purpose, but the enzyme has several demerits for cell experiments, including poor substrate solubility, possible conversion to dehydrogenase, and relatively high pH optimum [9]. However, the most serious point is the formation of urate, which scavenges OH Á and 1 O 2 [10].…”

“…However, the most serious point is the formation of urate, which scavenges OH Á and 1 O 2 [10]. We previously developed an O 2 À -generating nanodevice consisting of Nox2, phosphatidylinositol, and genetically engineered activating factors [9]. The device had high O 2 À -generating activity and stability at 37 1C in ordinary buffers.…”

An improved superoxide-generating nanodevice for oxidative stress studies in cultured cells

“…We previously developed an O 2 − -generating nanodevice based on Nox2 enzyme [22]. Nox2 was the first discovered Nox family enzyme in phagocytic cells, and subsequently found in many other types of cells.…”