Reagents and instruments for immunology, cell biology and molecular biology.

Dihydroethidium [104821-25-2]

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Cat# HY-D0079-10mg

Size : 10mg

Brand : MedChemExpress

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Description

Dihydroethidium, also known as DHE, is a peroxide indicator. Dihydroethidium penetrates cell membranes to form a fluorescent protein complex with blue fluoresces. After entering the cells, Dihydroethidium is mainly localized in the cell membrane, cytoplasm and nucleus, and the staining effect is the strongest in the nucleus. Dihydroethidium produces inherent blue fluorescence with a maximum excitation wavelength of 370 nm and a maximum emission wavelength of 420 nm; after dehydrogenation, Dihydroethidium combines with RNA or DNA to produce red fluorescence with a maximum excitation wavelength of 300 nm and a maximum emission wavelength of 610 nm. 535 nm can also be used as the excitation wavelength for actual observation^[1].

In Vitro

General Protocol
Preparation of Dihydroethidium working solution
1.1 Preparation of the stock solution
Dissolve 1 mg of Dihydroethidium in 0.31 mL of DMSO to obtain 10 mM of Dihydroethidium.
Note: It is recommended to store the stock solution at -20°C or -80°C away from light and avoid repetitive freeze-thaw cycles.
1.2 Preparation of Dihydroethidium working solution
Dilute the stock solution in serum-free cell culture medium or PBS to obtain 1-10 μM of Dihydroethidium working solution.
Note: Please adjust the concentration of Dihydroethidium working solution according to the actual situation.
Cell staining
2.1 For suspension cells: Centrifuge at 1000 g at 4°C for 3-5 minutes and then discard the supernatant. Wash twice with PBS, 5 minutes each time.
For adherent cells: Discard the cell culture medium, and add trypsin to dissociate cells to make a single-cell suspension. Centrifuge at 1000 g at 4°C for 3-5 minutes and then discard the supernatant. Wash twice with PBS, 5 minutes each time.
2.2 Add 1 mL of Dihydroethidium working solution, and then incubate at room temperature for 30 minutes.
2.3 Centrifuge at 400 g at 4°C for 3-4 minutes and then discard the supernatant.
2.4 Wash twice with PBS, 5 minutes each time.
2.5 Resuspend cells with serum-free cell culture medium or PBS, and then detect by fluorescence microscope or flow cytometer.

Storage
-20°C, 1 year
Protect from light

Precautions
1. It is recommended to store the stock solution at -20℃ or -80℃ away from light and avoid repetitive freeze-thaw cycles.
2. Please adjust the concentration of Dihydroethidium working solution according to the actual situation.
3. This product is for R&D use only, not for drug, household, or other uses.
4. For your safety and health, please wear a lab coat and disposable gloves to operate.

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

Solvent & Solubility

In Vitro:

DMSO : ≥ 100 mg/mL (317.05 mM; Hygroscopic DMSO has a significant impact on the solubility of product, please use newly opened DMSO)

*"≥" means soluble, but saturation unknown.

Preparing
Stock Solutions

Concentration Solvent Mass	1 mg	5 mg	10 mg

1 mM	3.1705 mL	15.8524 mL	31.7048 mL
5 mM	0.6341 mL	3.1705 mL	6.3410 mL
10 mM	0.3170 mL	1.5852 mL	3.1705 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 (protect from light). When stored at -80°C, please use it within 6 months. When stored at -20°C, please use it within 1 month.

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 40% PEG300 5% Tween-80 45% Saline
Solubility: 2.5 mg/mL (7.93 mM); Suspended solution; Need ultrasonic

This protocol yields a suspended solution of 2.5 mg/mL. Suspended solution can be used for oral and intraperitoneal injection.
Taking 1 mL working solution as an example, add 100 μL DMSO stock solution (25.0 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.
Protocol 2

Add each solvent one by one: 10% DMSO 90% (20% SBE-β-CD in Saline)
Solubility: 2.5 mg/mL (7.93 mM); Suspended solution; Need ultrasonic

This protocol yields a suspended solution of 2.5 mg/mL. Suspended solution can be used for oral and intraperitoneal injection.
Taking 1 mL working solution as an example, add 100 μL DMSO stock solution (25.0 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.