Dcfh-da itself has no fluorescence and can freely pass through the cell membrane. After entering the cell, DCFH can be hydrolyzed by the esterase in the cell. DCFH cannot permeate the cell membrane, making it easy for the probe to be loaded into the cell. Intracellular reactive oxygen species can oxidize non-fluorescent DCFH to produce fluorescent DCF. Detecting the fluorescence of DCF can determine the level of intracellular reactive oxygen species.

Operation steps (for reference only) :

1. Preparation of probe: 10mM (4.8729mg dissolved in 1mL DMSO)

2. Loading probe:

For cells with a short stimulation time (usually less than 2 hours), the probe is loaded first, and then the cells are stimulated with active oxygen positive controls or drugs of their own interest. For cells that are stimulated for a long time (usually more than 6 hours), the cells are first stimulated with active oxygen positive controls or drugs of interest to themselves, and then loaded with probes.

In situ probe loading: This method is only suitable for adherent cell culture. The final concentration of DCFH-DA was 10 μmol /L after dilution of 1 ∶1000 with serum-free medium. Remove the cell culture medium and add appropriate volume of diluted DCFH-DA. The volume added should be sufficient to cover the cells, and usually no less than 1mL of diluted DCFH-DA is added to one hole of the six-well plate. Incubate the cells at 37℃ for 20 minutes. The cells were washed three times with serum-free cell culture solution to fully remove DCFH-DA that did not enter the cells. Usually reactive oxygen species positive control can significantly increase reactive oxygen species levels after stimulating cells for 20 to 30 minutes.

After cell collection, the probe was loaded: DCFH-DA was diluted in serum-free medium at 1:1000 to a final concentration of 10μmol/L. After collection, the cells were suspended in diluted DCFH-DA at a cell concentration of 1 million to 20 million /mL, and incubated in a cell incubator at 37℃ for 20 minutes. Invert and mix every 3-5 minutes so that the probe and cells are fully in contact. The cells were washed three times with serum-free cell culture solution to fully remove DCFH-DA that did not enter the cells. Stimulate cells directly with active oxygen positive controls or drugs of interest, or divide cells into several parts to stimulate cells. Usually reactive oxygen species positive control can significantly increase reactive oxygen species levels after stimulating cells for 20 to 30 minutes.

Third, detection:

Samples loaded with probes in situ can be observed directly with a laser confocal microscope, or cells can be collected and examined with a fluorescence spectrophotometer, a fluorometer, or a flow cytometry. Samples loaded with probes after cell collection can be examined by fluorescence spectrophotometer, fluorescent enzyme spectrometer or flow cytometry, or can be directly observed by laser confocal microscopy.

4. Parameter setting

The excitation wavelength of 488nm and the emission wavelength of 525nm were used to detect the intensity of fluorescence before and after stimulation in real time or time point by point. The fluorescence spectrum of DCF is very similar to that of FITC, and DCF can be detected by FITC parameter Settings.

Note:

1. After the probe is loaded, it is necessary to wash the remaining probes that have not entered the cell, otherwise the background will be high.

2. After the probe is loaded and the residual probe is washed, the excitation wavelength and emission wavelength can be scanned to confirm whether the probe is properly loaded. The excitation and emission spectra of DCF are shown in the figure above.

3. Minimize the time from probe loading to determination (except stimulation time) to reduce possible errors.

4. For some cells, if it is found that the fluorescence of negative control cells without stimulation is also strong, DCFH-DA can be diluted according to 1:2000 ~ 1:5000, so that the concentration of DCFH-DA when loading the probe is 2-5 μmol/L. The loading time of the probe can also be adjusted appropriately within 15 to 60 minutes according to the situation.

5. For your safety and health, please wear a lab coat and disposable gloves.

Examples of using this product（for reference only）

In Vitro:

Cell（HK-2 human kidney proximal tubular cells，20 μmol/L DCFH-DA，37℃ for 30 min）:

Cells grown on coverslips were incubated with 20 μmol/L 2′,7′-dichlorofluorescin diacetate at 37 ℃ for 30 min in the dark and then were washed with phosphate buffer saline. Cells were fixed in 4% paraformaldehyde, counterstained with DAPI, then washed 3 times with phosphate buffer saline. Cells were mounted with an anti-fading mounting medium and fluorescence intensity was detected by a fluorescence microscope with an excitation wavelength at 488 nm and an emission wavelength at 525 nm.

Reference：

Li S, Zheng L, Zhang J, Liu X, Wu Z. Inhibition of ferroptosis by up-regulating Nrf2 delayed the progression of diabetic nephropathy. Free Radic Biol Med. 2021 Jan;162:435-449. doi: 10.1016/j.freeradbiomed.2020.10.323. Epub 2020 Nov 2. PMID: 33152439.

Cell（vascular smooth muscle cells/VSMCs; 10 μM DCFH- DA; 37℃,30 min）:

The intracellular ROS content was measured with the fluorescent probe DCFH- DA(Solarbio, China), which itself is nonfluorescent. After freely passing through the cell membrane, Tt is hydrolyzed to impenetrable DCFH and remains inside the cells. Intracellular ROS oxidizes DCFH to fluorescent DCF, which indicates the intracellular ROS levels according to the fluorescence intensity. VSMCs with five different treatments were incubated with serum-free DMEM with a DCFH-DA final concentration of 10 uM for 30 min at 37 ℃. Next, we washed the VSMC-DCFH-DA mixture with serum-free DMEM three times and resuspended it in 1× PBS solution. The prepared VSMC samples were analyzed using flow cytometry.

Reference：

Shao S, Liu Y, Hong W, Mo Y, Shu F, Jiang L, Tan N. Influence of FOSL1 Inhibition on Vascular Calcification and ROS Generation through Ferroptosis via P53-SLC7A11 Axis. Biomedicines. 2023 Feb 20;11(2):635. doi: 10.3390/biomedicines11020635. PMID: 36831172; PMCID: PMC9953509.

Cell（myoblasts；10 μM DCFH-DA，37℃，20 min）:

The myoblasts were cultured in the 35 mm cell culture dishes, and after heat stress, the cells were incubated with 10 μM dichlorofluorescein diacetate (DCFH-DA) (Solarbio, Beijing,China) at 37℃ for 20 min, after washing three times by DMEM/F12 to remove the extracellular DCFH-DA of myoblasts, the laser scanning confocal microscope was used to obtain the ROS images.

Reference：

Lu J, Li H, Yu D, Zhao P, Liu Y. Heat stress inhibits the proliferation and differentiation of myoblasts and is associated with damage to mitochondria. Front Cell Dev Biol. 2023 Apr 11;11:1171506. doi: 10.3389/fcell.2023.1171506. PMID: 37113771; PMCID: PMC10126414.

Cell（PC12 cells；10 μM DCFH-DA ；37 ℃，10 min）:

The cells (1×10⁴/well) were seeded in 96-well plates, and were treated with various concentrations of baicalein (40, 60 and 80 μM) for 2 h, followed by incubation with Aβ25–35 (20 μM) for 24 h. Then, the cells were incubated with 10 μM DCFH-DA and 10 μM Hoechst 33342 for 10 min at 37 ℃ in the darkness, and washed three times with PBS. The fluorescence intensity was detected and the images were photographed using a Cytation imaging reader. The intracellular ROS levels were indicated by total fluorescence intensity.

Reference：

Gao L, Zhou F, Wang KX, Zhou YZ, Du GH, Qin XM. Baicalein protects PC12 cells from Aβ25-35-induced cytotoxicity via inhibition of apoptosis and metabolic disorders. Life Sci. 2020 May 1;248:117471. doi: 10.1016/j.lfs.2020.117471. Epub 2020 Feb 26. PMID: 32112868.