Improved detection of nitric oxide radical (NO^•) production in an activated macrophage culture with a radical scavenger, car☐y PTIO, and Griess reagent

Abstract

An improved method for the detection of nitric oxide radicals (NO'v in cultures of activated macrophages was developed, involving a nitric oxide radical scavenger, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-3-oxide-l-oxyl (carboxy PTIO) and Griess reagent. A murine macrophage-like cell line, J774.1, was activated with interferon-y (IFN-T) and bacterial lipopolysaccharide (LPS), which induced the production and secretion of NO~ into the culture supernatant. Addition of carboxy PTIO to the activated macrophages increased the amount of NO~ to 4-to 5-fold without cell damages, probably because carboxy PTIO rapidly reacted with NO" to form NO~, which was finally assayed by the Griess reaction.Key words: Murine macrophage-like cell; Nitric oxide; Macrophage activation; Carboxy PTIO activated macrophages, we tried to use carboxy PTIO as an oxidizing reagent for NO', which directly oxidizes NO" to form NO~ [7], which readily reacts with another NO" to form N203 [NO~ + NO" ~--N203], and then N203 reacts with H20 to generate NO2 [N203 + H20 ~-2NO~ + 2H +] [8]. As a whole, carboxy PTIO mediates the conversion of NO" to NO2 in a molar to a molar ratio [7]. The resultant product, NO2, of NO" and carboxy PTIO, together with NO2 automatically produced from NO" in biological systems (see Fig. 1 for a model) should be detected by the Griess reagent. In this paper, we present results showing that activated macrophages produce 4-to 5-fold higher levels of NO~ in the presence of carboxy PTIO than in its absence, and suggest that the addition of carboxy PTIO to macrophage cultures will improve detection of NO" produced by activated macrophages. Materials and methods

Nitrososphaera viennensis gen. nov., sp. nov., an aerobic and mesophilic, ammonia-oxidizing archaeon from soil and a member of the archaeal phylum Thaumarchaeota

“…Genes for urea utilization were recently also found in metagenomic analyses of marine group I thaumarchaeotes from Arctic and meso-/bathypelagic waters and marine sediments, indicating that urea is a more widespread energy (and perhaps also carbon) source for AOA (Alonso-Sáez et al , 2012; Park et al , 2012) than was previously appreciated. The NO scavenger carboxy-PTIO [2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide] (Amano & Noda, 1995) has been shown to inhibit growth and nitrite production of both strain EN76 T (Shen et al , 2013) and ‘ Candidatus Nitrosopumilus maritimus’ SCM1 (Yan et al , 2012), indicating an important role for NO in their energy metabolism. In addition, hydroxylamine is probably an intermediate during the oxidation of ammonia to nitrite, as shown for AOB [Arp & Stein (2003) and references therein; Hooper & Terry (1979)], and recently also shown for the marine AOA ‘ Candidatus Nitrosopumilus maritimus’ SCM1 (Vajrala et al , 2013).…”

α 1-antitrypsin modulates microglial-mediated neuroinflammation and protects microglial cells from amyloid-β-induced toxicity

“…Accumulated nitrite, a stable oxidation product of nitric oxide (NO), was measured using Griess reagents [21]. For this measurement, 50 μl of primary microglial cell supernatants was transferred to a 96-well microtiter plate, and 50 μl of solution 1 (1% sulfanilamide in 5% phosphoric acid) was added.…”

Heat shock protein 60: an endogenous inducer of dopaminergic cell death in Parkinson disease

“…Accumulated nitrite, a stable oxidation product of NO, was measured using Griess reagents [36]. Fifty microliters of primary microglial cell supernatants were transferred to a 96-well microtiter plate and 50 μl of solution 1 (1% sulfanilamide in 5% phosphoric acid) were added.…”