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  4. 2,7-Dichlorodihydrofluorescein

2,7-Dichlorodihydrofluorescein  (Synonyms: DCFH2)

Cat. No.: HY-153006 Purity: 99.14%
SDS
COA
Handling Instructions Technical Support

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).

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2,7-Dichlorodihydrofluorescein Chemical Structure

2,7-Dichlorodihydrofluorescein Chemical Structure

CAS No. : 106070-31-9

Size Price Stock Quantity
Solid + Solvent (Highly Recommended)
10 mM * 1 mL in DMSO
ready for reconstitution
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10 mM * 1 mL in DMSO In-stock
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Based on 5 publication(s) in Google Scholar

2,7-Dichlorodihydrofluorescein Related Antibodies

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Description

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].

In Vitro

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.

2,7-Dichlorodihydrofluorescein Related Antibodies
Molecular Weight

403.21

Formula

C20H12Cl2O5
CAS No.
Appearance

Solid

Color

Pink to orange

SMILES

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

Room temperature in continental US; may vary elsewhere.
Storage
Powder -20°C 3 years
4°C 2 years
In solvent -80°C 6 months
-20°C 1 month
Solvent & Solubility
In Vitro: 

DMSO : 250 mg/mL (620.02 mM; Need ultrasonic; Hygroscopic DMSO has a significant impact on the solubility of product, please use newly opened DMSO)

Preparing
Stock Solutions
Concentration Solvent Mass 1 mg 5 mg 10 mg
1 mM 2.4801 mL 12.4005 mL 24.8010 mL
5 mM 0.4960 mL 2.4801 mL 4.9602 mL
View the Complete Stock Solution Preparation Table

* 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.

  • Molarity Calculator

  • Dilution Calculator

Mass (g) = Concentration (mol/L) × Volume (L) × Molecular Weight (g/mol)

Mass
=
Concentration
×
Volume
×
Molecular Weight *

Concentration (start) × Volume (start) = Concentration (final) × Volume (final)

This equation is commonly abbreviated as: C1V1 = C2V2

Concentration (start)

C1

×
Volume (start)

V1

=
Concentration (final)

C2

×
Volume (final)

V2

In Vivo:

Select the appropriate dissolution method based on your experimental animal and administration route.

For the following dissolution methods, please ensure to first prepare a clear stock solution using an In Vitro approach and then sequentially add co-solvents:
To ensure reliable experimental results, the clarified stock solution can be appropriately stored based on storage conditions. As for the working solution for in vivo experiments, it is recommended to prepare freshly and use it on the same day.
The percentages shown for the solvents indicate their volumetric ratio in the final prepared solution. If precipitation or phase separation occurs during preparation, heat and/or sonication can be used to aid dissolution.

  • Protocol 1

    Add each solvent one by one:  10% DMSO    90% (20% SBE-β-CD in Saline)

    Solubility: ≥ 20.83 mg/mL (51.66 mM); Clear solution

    This protocol yields a clear solution of ≥ 20.83 mg/mL (saturation unknown).

    Taking 1 mL working solution as an example, add 100 μL DMSO stock solution (208.3 mg/mL) to 900 μL 20% SBE-β-CD in Saline, and mix evenly.

    Preparation of 20% SBE-β-CD in Saline (4°C, storage for one week): 2 g SBE-β-CD powder is dissolved in 10 mL Saline, completely dissolve until clear.
  • Protocol 2

    Add each solvent one by one:  10% DMSO    40% PEG300    5% Tween-80    45% Saline

    Solubility: ≥ 2.08 mg/mL (5.16 mM); Clear solution

    This protocol yields a clear solution of ≥ 2.08 mg/mL (saturation unknown).

    Taking 1 mL working solution as an example, add 100 μL DMSO stock solution (20.8 mg/mL) to 400 μL PEG300, and mix evenly; then add 50 μL Tween-80 and mix evenly; then add 450 μL Saline to adjust the volume to 1 mL.

    Preparation of Saline: Dissolve 0.9 g sodium chloride in ddH₂O and dilute to 100 mL to obtain a clear Saline solution.
In Vivo Dissolution Calculator
Please enter the basic information of animal experiments:

Dosage

mg/kg

Animal weight
(per animal)

g

Dosing volume
(per animal)

μL

Number of animals

Recommended: Prepare an additional quantity of animals to account for potential losses during experiments.
Please enter your animal formula composition:
%
DMSO +
% 
+
%
Tween-80 +
%
Saline
Recommended: Keep the proportion of DMSO in working solution below 2% if your animal is weak.
The co-solvents required include: DMSO, . All of co-solvents are available by MedChemExpress (MCE). , Tween 80. All of co-solvents are available by MedChemExpress (MCE).
Calculation results:
Working solution concentration: mg/mL
Method for preparing stock solution: mg drug dissolved in μL  DMSO (Stock solution concentration: mg/mL).
The concentration of the stock solution you require exceeds the measured solubility. The following solution is for reference only. If necessary, please contact MedChemExpress (MCE).
Method for preparing in vivo working solution for animal experiments: Take μL DMSO stock solution, add μL . μL , mix evenly, next add μL Tween 80, mix evenly, then add μL Saline.
 If the continuous dosing period exceeds half a month, please choose this protocol carefully.
Please ensure that the stock solution in the first step is dissolved to a clear state, and add co-solvents in sequence. You can use ultrasonic heating (ultrasonic cleaner, recommended frequency 20-40 kHz), vortexing, etc. to assist dissolution.
Purity & Documentation

Purity: 99.14%

SDS (392 KB)

COA (254 KB) HNMR (264 KB) LCMS (98 KB)

Handling Instructions (2659 KB)
References

Complete Stock Solution Preparation Table

* 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.

Optional Solvent Concentration Solvent Mass 1 mg 5 mg 10 mg 25 mg
DMSO 1 mM 2.4801 mL 12.4005 mL 24.8010 mL 62.0024 mL
5 mM 0.4960 mL 2.4801 mL 4.9602 mL 12.4005 mL
10 mM 0.2480 mL 1.2400 mL 2.4801 mL 6.2002 mL
15 mM 0.1653 mL 0.8267 mL 1.6534 mL 4.1335 mL
20 mM 0.1240 mL 0.6200 mL 1.2400 mL 3.1001 mL
25 mM 0.0992 mL 0.4960 mL 0.9920 mL 2.4801 mL
30 mM 0.0827 mL 0.4133 mL 0.8267 mL 2.0667 mL
40 mM 0.0620 mL 0.3100 mL 0.6200 mL 1.5501 mL
50 mM 0.0496 mL 0.2480 mL 0.4960 mL 1.2400 mL
60 mM 0.0413 mL 0.2067 mL 0.4133 mL 1.0334 mL
80 mM 0.0310 mL 0.1550 mL 0.3100 mL 0.7750 mL
100 mM 0.0248 mL 0.1240 mL 0.2480 mL 0.6200 mL
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2,7-Dichlorodihydrofluorescein Related Classifications

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2,7-Dichlorodihydrofluorescein106070-31-9DCFH2DCFH 2DCFH-2Biochemical Assay ReagentsInhibitorinhibitorinhibit

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