| Description | H2DCFDA (DCFH-DA) belongs to the class of green fluorescent dyes and is a probe for the detection of intracellular reactive oxygen species (ROS) (Ex/Em=488/525 nm) with cell membrane permeability. |
| In vitro | 方法：Flow cytometry 检测 ROS 水平：1、将 H2DCFDA 溶解为 10 mM 的 DMSO 原液，并在使用前用 PBS 进一步稀释。2、将粘附细胞与 5 µM H2DCFDA 溶液在 37℃ 下避光孵育 30 min，然后用 0.05% trypsin-EDTA 溶液收获，悬浮在新鲜培养基中，并立即用流式细胞仪 (488 nm) 进行分析。[1]方法：Confocal microscopy 检测 ROS 水平：1、将 H2DCFDA 溶解为 10 mM 的 DMSO 原液，并在使用前用 PBS 进一步稀释。2、将含有细胞的盖玻片置于 5 µM H2DCFDA 染色溶液中，在 37℃ 下避光孵育 60 min，然后清洗并用配备有氩激光器的共焦激光扫描显微镜 Leica TCS SL 成像。[1] |
| In vivo | 方法：fluorescence microscope 分析 LPS 诱导腹膜炎小鼠的氧化活性：1、将 H2DCFDA 溶解在 100 µL 乙醇中，并在使用前用 PBS 进一步稀释。2、C57BL/6J 小鼠腹腔注射 LPS (0.1-1mg/mL) 诱导腹膜炎。3.5 h 后，腹腔注射 H2DCFDA (0.1-0.8 mg/ml)。3、H2DCFDA 注射后 30 min，颈椎脱臼处死动物，用 pH 7.4 的 5 mL 冰冷 HBSS 溶液冲洗，回收腹膜细胞。4、腹膜细胞用 PBS 洗涤并重悬。在 37℃ 聚苯乙烯培养皿中孵育 30 min，用粘附法除去巨噬细胞。回收上清液，用细胞离心器将大约 20000-25000 个白细胞加到显微镜载玻片上。然后使用荧光显微镜对载玻片进行分析。[2] |
| Synonyms | 2',7'-二氯荧光素二乙酸酯, 2',7'-Dichlorodihydrofluorescein diacetate, DCFH-DA |
| molecular weight | 487.29 |
| Molecular formula | C24H16Cl2O7 |
| CAS | 4091-99-0 |
| Storage | keep away from direct sunlight | Powder: -20°C for 3 years | In solvent: -80°C for 1 year |
| Solubility | Ethanol: 14.29 mg/mL (29.33 mM), Sonication is recommended. DMSO: 50 mg/mL (102.61 mM) |
| References | 1. Lyublinskaya OG, et al. Redox environment in stem and differentiated cells: A quantitative approach. Redox Biol. 2017 Aug;12:758-769. 2. Linda S, et al. Method for analysis of intracellular production of free radicals in vivo. SWEDISH DEFENCE RESEARCH AGENCY. 2001 Mar. |
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