Fernandes Denise C, Gonçalves Renata C, Laurindo Francisco R M
Vascular Biology Laboratory, Heart Institute (InCor), University of São Paulo School of Medicine, Av. Eneas Carvalho Aguiar, 44, Annex II, 9th Floor, CEP 05403-000, São Paulo, Brazil.
Methods Mol Biol. 2017;1527:233-249. doi: 10.1007/978-1-4939-6625-7_19.
The fluorogenic probe dihydroethidium (DHE) is widely used for detecting intracellular superoxide. DHE oxidation by superoxide generates specifically the compound 2-hydroxyethidium (2-EOH), so that 2-EOH detection confers specificity to superoxide assessment among many other reactive oxygen species. However, DHE oxidation in biological systems leads to formation of other fluorescent products, particularly ethidium, usually formed at higher quantities than 2-EOH. Since both 2-EOH and ethidium are fluorescent, their identification and quantification is possible only after their physical separation by HPLC. Here we describe the detailed procedures for superoxide measurement in cells (adhered or not) and fresh tissues fragments, followed by acetonitrile extraction and simultaneous fluorescent detection of 2-EOH and ethidium and absorbance detection of remaining unreacted DHE. In addition we report the use of DHE/HPLC for measuring NADPH oxidase activity in enriched-membrane fraction isolated from cells or tissues. These methods can improve accuracy and precision of quantitative superoxide measurements in biological samples.
荧光探针二氢乙锭（DHE）被广泛用于检测细胞内的超氧化物。超氧化物将DHE氧化会特异性地生成化合物2-羟基乙锭（2-EOH），因此检测2-EOH可在众多其他活性氧物种中赋予超氧化物评估特异性。然而，生物系统中的DHE氧化会导致形成其他荧光产物，尤其是乙锭，其生成量通常高于2-EOH。由于2-EOH和乙锭都具有荧光性，只有通过高效液相色谱（HPLC）对它们进行物理分离后，才能对其进行鉴定和定量。在此，我们描述了在细胞（贴壁或未贴壁）和新鲜组织碎片中测量超氧化物的详细步骤，随后进行乙腈萃取，并同时对2-EOH和乙锭进行荧光检测以及对剩余未反应的DHE进行吸光度检测。此外，我们报告了使用DHE/HPLC来测量从细胞或组织中分离出的富集膜组分中的NADPH氧化酶活性。这些方法可以提高生物样品中超氧化物定量测量的准确性和精密度。
