二氢罗丹明123 (DHR123)是一种不带电荷且无荧光的活性氧 (ROS)指示剂，可以跨膜扩散，进入细胞后如果有氧自由基，它在线粒体中被氧化成罗丹明123阳离子，呈现绿色荧光 (Ex/Em=507/529 nm)。DHR123常用来检测内皮细胞、嗜酸性细胞和反应性小胶质细胞产生的活性氧中间物。
染色程序：
(1) 用培养基稀释5mM DHR123 溶液，稀释50倍得到100 µM DHR123工作液。
(2) 准备两批HL60细胞，细胞密度～6×10⁶ 个/mL缓冲溶液为Krebs-Ringer buffer.
(3) 将HL60细胞十分之一体积的DHR123工作液加入两批HL60细胞中，DHR123终浓度为10 µM，并在37 ℃下孵育30分钟。
(4) 其中一批HL60细胞与DHR123培养后，加入氧自由基诱导剂phorbol 12-myristate 13-acetat (PMA)终浓度50 µM。
(5) 另一批HL60细胞加入SNARF-1 AM acetate (SNARF-AM)探针终浓度5 µM在37°C培养10 min，离心用并用培养液洗涤细胞。
(6) 用带有荧光素滤光片的荧光显微镜观察细胞。
注意事项：
(1) 溶液现配现用。
(2) 为了您的安全和健康，请穿实验服并戴一次性手套。
参考文献：
Lydia M. Henderson et al. Dihydrorhodamine 123: a fluorescent probe for superoxide generation. Eur.J.Biochem. 217, 973-980.
Product Description
Dihydrorhodamine 123 (DHR123) is a non-charged and non-fluorescent reactive oxygen species (ROS) indicator that can diffuse across cell membranes. After entering cells, in the presence of oxygen free radicals, it is oxidized to rhodamine 123 cation in mitochondria and emits green fluorescence (Ex/Em= 507/529 nm). DHR123 is commonly used to detect reactive oxygen intermediates produced by endothelial cells, eosinophils, and reactive microglia.
Staining Procedure
(1) Dilute the 5 mM DHR123 stock solution 50-fold with culture medium to obtain a 100 µM DHR123 working solution.
(2) Prepare two batches of HL60 cells at a cell density of ~6×10⁶ cells/mL, using Krebs-Ringer buffer as the buffer system.
(3) Add DHR123 working solution at 1/10 the volume of HL60 cell suspension to both batches, yielding a final DHR123 concentration of 10 µM, and incubate at 37 ℃ for 30 minutes.
(4) To one batch of HL60 cells after incubation with DHR123, add the oxygen free radical inducer phorbol 12-myristate 13-acetate (PMA) to a final concentration of 50 µM.
(5) To the other batch of HL60 cells, add SNARF-1 AM acetate (SNARF-AM) probe to a final concentration of 5 µM, incubate at 37 ℃ for 10 min, centrifuge, and wash the cells with culture medium.
(6) Observe the cells under a fluorescence microscope equipped with a fluorescein filter set.
Precautions
(1) Prepare solutions fresh before use.
(2) For your safety and health, please wear a lab coat and disposable gloves.
Reference
Lydia M. Henderson et al. Dihydrorhodamine 123: a fluorescent probe for superoxide generation. Eur. J. Biochem. 217, 973-980.
ZJ26F0737184
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