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Reagenzien und Instrumente für die Immunologie Zellbiologie und Molekularbiologie

H2DCFDA [4091-99-0]

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Katalog-Nummer HY-D0940-50mg

Size : 50mg

Contact local distributor :

Telefonnummer : +1 850 650 7790

Marke : MedChemExpress

Weitere Informationen anfordern

Contact local distributor :

Telefonnummer : +1 850 650 7790

Beschreibung

H2DCFDA (DCFH-DA) is a cell-permeable probe used to detect intracellular reactive oxygen species (ROS) (Ex/Em=488/525 nm)^[1].

In Vitro

Guide (The following is our recommended protocol. This protocol is only a guide and should be modified according to your specific needs).
1.1 Preparation of stock solution
Use anhydrous DMSO to prepare a 10 mM H2DCFDA stock solution.
Note: It is recommended that the H2DCFDA stock solution be stored at -20 ℃ or -80 ℃ in the dark after aliquoting.
1.2 Preparation of working solution
Use preheated serum-free cell culture medium or PBS to dilute the stock solution to prepare a 1-10 μM H2DCFDA working solution.
Note: Please adjust the concentration of H2DCFDA working solution according to actual conditions and prepare it before use.
2. Cell staining (suspended cells)
2.1 Collect cells by centrifugation and wash twice with PBS for 5 minutes each time. The cell density is 1×10⁶/mL.
2.2 Add 1 mL of dye working solution and incubate at room temperature for 5-30 minutes.
2.3 Centrifuge at 400 g for 3-4 minutes and discard the supernatant.
2.4 Add PBS to wash the cells twice, 5 minutes each time.
2.5 Resuspend the cells in 1 mL of serum-free medium or PBS and observe using a fluorescence microscope or flow cytometer.
3. Cell staining (adherent cells)
3.1 Culture the adherent cells on a sterile coverslip.
3.2 Remove the coverslip from the culture medium and remove the excess culture medium.
3.3 Add 100 μL of dye working solution, gently shake to completely cover the cells, and incubate for 5-30 minutes.
3.4 Aspirate the dye working solution, wash 2-3 times with culture medium, 5 minutes each time, and observe using a fluorescence microscope or flow cytometer.
Note: If flow cytometry is required, the cells need to be digested with trypsin and resuspended before staining.

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

Lösungsmittel & Löslichkeit

In Vitro:

DMSO : 125 mg/mL (256.52 mM; Need ultrasonic; Hygroscopic DMSO has a significant impact on the solubility of product, please use newly opened DMSO)

Ethanol : 20 mg/mL (41.04 mM; Need ultrasonic)

Preparing
Stock Solutions

Concentration Solvent Mass	1 mg	5 mg	10 mg

1 mM	2.0522 mL	10.2608 mL	20.5217 mL
5 mM	0.4104 mL	2.0522 mL	4.1043 mL
10 mM	0.2052 mL	1.0261 mL	2.0522 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: 2.08 mg/mL (4.27 mM); Suspended solution; Need ultrasonic

This protocol yields a suspended solution of 2.08 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 (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.
Protocol 2

Add each solvent one by one: 10% DMSO 90% (20% SBE-β-CD in Saline)
Solubility: ≥ 2.08 mg/mL (4.27 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 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 3

Add each solvent one by one: 10% DMSO 90% Corn Oil
Solubility: ≥ 2.08 mg/mL (4.27 mM); Clear solution

This protocol yields a clear solution of ≥ 2.08 mg/mL (saturation unknown). If the continuous dosing period exceeds half a month, please choose this protocol carefully.
Taking 1 mL working solution as an example, add 100 μL DMSO stock solution (20.8 mg/mL) to 900 μL Corn oil, and mix evenly.