2,7-Dichlorodihydrofluorescein  (Synonyms: DCFH2)

2,7-Dichlorodihydrofluorescein (DCFH2) is a non-fluorescent reactive oxygen species (ROS) probe with an excitation wavelength of 485-500 nm and an emission wavelength of 515-530 nm. 2,7-Dichlorodihydrofluorescein is first hydrolyzed by intracellular esterases and then oxidized by ROS to generate non-biomembrane-permeable, highly fluorescent 2,7-dichlorofluorescein (DCF). The fluorescence intensity of DCF is positively correlated with the ROS concentration. 2,7-Dichlorodihydrofluorescein can undergo oxidation reactions with a variety of ROS (such as ·OH, H₂O₂, ONOO^-, etc.) and is used to quantitatively detect the level of oxidative stress inside and outside cells. It is suitable for oxidative stress analysis in in vitro cell models and in vivo targeted delivery (such as liver-targeted liposomes)^[1][2].

2,7-Dichlorodihydrofluorescein (DCFH2) Protocol^[1][2]
1. Preparation of working solution
2,7-Dichlorodihydrofluorescein should be prepared immediately. Prepare a 10 μM working solution before use.
2. Cell/tissue treatment
In vitro experiments:
After culturing cells (such as HEPG2 and RAW 264.7 macrophages), add 10 μM 2,7-Dichlorodihydrofluorescein working solution (freshly prepared, protected from light) and incubate at 37°C for 30 minutes to 1 hour to ensure that 2,7-Dichlorodihydrofluorescein enters the cells through the cell membrane.
In vivo experiments:
5(6)-carboxyl-DCFH₂ (CDCFH₂) was encapsulated in liver-targeted liposomes and injected into mice through the tail vein for real-time fluorescence imaging in a liver ischemia-reperfusion model to detect oxidative stress in hepatocytes.
Positive control:
oxidants (such as PMA, H₂O₂) were added to induce ROS generation; negative control: antioxidants (such as SOD, CAT) or solvents (DMSO) were added.
Control of interference factors: Avoid strong light exposure, and the incubation time needs to be optimized in advance to ensure that esterase activity does not affect the results.
3. Detection and application
Fluorescence spectroscopy:
The excitation wavelength of DCF is 485-500 nm, and the emission wavelength is 515-530 nm. The fluorescence intensity of DCF in cell lysate or tissue homogenate was detected to quantify the ROS level.
Flow cytometry:
After single cell suspension is stained with 2,7-Dichlorodihydrofluorescein, flow cytometry can be used to detect intracellular fluorescence intensity and analyze differences in oxidative stress in cell populations.
Fluorescence microscopy/confocal imaging:
After staining living cells or tissue sections, observe DCF fluorescence localization (such as cell membrane, cytoplasm), and combine with Lysotracker (HY-D1300) and other markers for co-localization analysis.
4. Precautions
Light-proof operation:
All steps must be performed in the dark to avoid photooxidation of DCFH2.
Fresh preparation:
The working solution should be prepared and used immediately, and the storage solution should be stored in the dark for a short period of time (≤4 weeks) after aliquoting.
Cell specificity:
The difference in esterase activity of different cells affects the hydrolysis efficiency of DCFH2-DA, and preliminary experiments are required to determine the optimal loading concentration (10-20 μM) and time.
Interference elimination:
The concentration of solvent (such as DMSO) should be ≤0.1% to avoid competitive inhibition of ROS reaction; enzyme inhibitors such as SOD and CAT are used to verify the oxidation pathway.

MedChemExpress (MCE) has not independently confirmed the accuracy of these methods. They are for reference only.

403.21

C₂₀H₁₂Cl₂O₅

106070-31-9

Solid

Pink to orange

O=C(C1=C(C2C3=CC(Cl)=C(C=C3OC4=CC(O)=C(C=C42)Cl)O)C=CC=C1)O

Room temperature in continental US; may vary elsewhere.

* Please refer to the solubility information to select the appropriate solvent. Once prepared, please aliquot and store the solution to prevent product inactivation from repeated freeze-thaw cycles.
Storage method and period of stock solution: -80°C, 6 months; -20°C, 1 month. When stored at -80°C, please use it within 6 months. When stored at -20°C, please use it within 1 month.

• [1]. Reiniers MJ, et al. Preparation and Practical Applications of 2',7'-Dichlorodihydrofluorescein in Redox Assays. Anal Chem. 2017 Apr 4;89(7):3853-3857.  [Content Brief]

  [2]. Chen X, et al. 2',7'-Dichlorodihydrofluorescein as a fluorescent probe for reactive oxygen species measurement: Forty years of application and controversy. Free Radic Res. 2010 Jun;44(6):587-604.  [Content Brief]