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• H2DCFDA (2′,7′-dichlorodihydrofluorescein diacetate)

H2DCFDA (2′,7′-dichlorodihydrofluorescein diacetate)

H2DCFDA (2′,7′-dichlorodihydrofluorescein diacetate) is a common reagent used for investigating the production of reactive oxygen species in living cells.

H2DCFDA is a non-fluorescent fluorescein derivative (its reduced acetylated form). The reagent begins to emit fluorescence only after cleavage of acetyl groups and the reagent’s oxidation in the cell while converting to 2’,7’-dichlorofluorescein. This is a bright green-fluorescent dye (absorption maximum 511 nm, fluorescence maximum 533 nm). This reagent can be used for assays in living cells and is not compatible with sample fixation.

Acetyl groups in H2DCFDA increase its lipophilicity and improve its cell membrane permeability. Once got into the cell, the dye is deacetylated by cell esterases, thus becoming charged and better fixed inside the cell. Oxidation with reactive oxygen forms results in the formation of a fluorescent product (2’,7’-dichlorofluorescein) and can be detected using various methods, for example with a flow cytometer, plate reader, or fluorescent microscope.

H2DCFDA（2′,7′-二氯二氢荧光素二乙酸酯）是一种常用试剂，用于研究活细胞中活性氧的产生。

H2DCFDA 是一种非荧光荧光素衍生物（其还原乙酰化形式）。该试剂仅在乙酰基裂解和试剂在细胞中氧化并转化为 2′,7′-二氯荧光素后才开始发射荧光。这是一种亮绿色荧光染料（最大吸收 511 nm，最大荧光 533 nm）。该试剂可用于活细胞检测，与样品固定不兼容。

H2DCFDA 中的乙酰基会增加其亲脂性并改善其细胞膜通透性。一旦进入细胞，染料就会被细胞酯酶脱乙酰化，从而带电并更好地固定在细胞内。用活性氧形式氧化会形成荧光产物（2′,7′-二氯荧光素），可以使用各种方法检测，例如使用流式细胞仪、读板器或荧光显微镜。

Recommendations for using the reagent使用试剂的建议：:

• Use a freshly prepared solution of the reagent (the working solution is not intended for long-term storage because of gradual reagent oxidation).
• Select an optimal working concentration of the reagent and incubation time required for reagent deacetylation and oxidation for the specific cell line and assay conditions. If there are no protocols recommended for the specific cell line, start with a concentration from 1 to 10 µM and incubation for 30 min.
• Do not incubate the dye with the cells in the presence of serum because it contains enzymes that cleave H2DCFDA.
• 使用新配制的试剂溶液（工作溶液不适合长期储存，因为试剂会逐渐氧化）。
  针对特定细胞系和测定条件，选择试剂脱乙酰和氧化所需的最佳试剂工作浓度和孵育时间。 如果没有针对特定细胞系推荐的方案，请从 1 到 10 µM 的浓度开始并孵育 30 分钟。
  不要在存在血清的情况下将染料与细胞一起孵育，因为它含有切割 H2DCFDA 的酶。