2,7-Dichlorodihydrofluorescein

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.