ROS Probes-All Eyes Are Focused Here！
This article explains the protocol to use different ROS probes.
H2DCFDA is a chemically reduced form of fluorescein used as an indicator for reactive oxygen species (ROS) in cells which has good cell membrane permeability, for example, to detect the generation of reactive oxygen intermediates in neutrophils and macrophages. Upon cleavage of the acetate groups by intracellular esterases and oxidation, the non-fluorescent H2DCFDA is converted to a highly fluorescent 2',7'-dichlorofluorescein (DCF) {Ex/Em: 488/525 nm}.
HKSOX-1 is a fluorescent probe for imaging and detection of endogenous superoxide anion radical (?O2?) in living cells and in?in vivo?experiments. It has excellent selectivity and sensitivity to superoxide anion radical. HKSOX-1 reacts with ?O2??in potassium phosphate buffer at 25 °C to produce an intense time-dependent fluorescence.
HKSOX-1r is also a fluorescent probe for imaging and detection of endogenous superoxide anion radical (?O2?) in living cells and in?in vivo?experiments. It is prepared from HKSOX-1R and it has better cellular permeability and retention.
HKPerox-2 is a highly selective and sensitive green fluorescent probe. Its selectivity for H2O2?is 30 times higher than that of other tested ROS/RNS. HKPerox-2 is an “O-methyl rhodol derivative”, which specifically recognizes H2O2?through Payne/dakin series reaction.
Steps for using H2DCFDA
Preparation of H2DCFDA working solution
1. Preparation of the stock solution
Dissolve 5 mg of H2DCFDA in 1.026 mL of DMSO to obtain 10 mM of H2DCFDA. (Note: It is recommended to store the stock solution at -20 °C or -80 °C. Protect from both light and air, and avoid freeze-thaw cycles.)
2. Preparation of H2DCFDA working solution
Dilute the stock solution in serum-free cell culture medium or PBS to obtain 5-10 μM of H2DCFDA working solution. (Note: Working solutions should be freshly prepared.)
Adjust the concentration of the H2DCFDA working solution according to the experimental requirements.
Staining of Suspension Cells
a. In 6-well culture plates, inoculate the appropriate amount of cells and incubate at 37 °C overnight.
b. After the cells’ density reaches 80-90%, start positive/negative control treatment (optional).
c. Negative control (optional): Wash cells twice with 1 mL PBS, add fresh 5 mM N-Acetylcysteine, and incubate at 37 °C for 1 h.
d. Discard the culture medium from the wells and add 1 mL of H2DCFDA working solution and then incubate at room temperature for 30 minutes.
e. Centrifuge at 400 g and at 4 °C for 3-4 minutes and then remove the supernatant carefully.
f. Wash the cells twice with PBS.
g. Resuspend cells with serum-free cell culture medium or PBS, and then detect H202?by fluorescence microscope or flow cytometer.
Staining of Adherent Cells
Adherent cells can be treated in the same way as suspension cells after trypsinization and re-suspension.
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Case Study
The following content is the case study related to the use of H2DCFDA?in vitro
Mammalian Cell Staining
A study, “Photodynamic therapy induces autophagy-mediated cell death in human colorectal cancer cells via activation of the ROS/JNK signaling pathway” proved that “m-THPC-PDT” or “VP-PDT” treatment significantly induced apoptosis in HCT116 cells with H2DCFDA staining and subsequent detection of H202?by flow cytometry. As shown in figure 2, ROS production increased in HCT116 cells in a time-dependent manner[1].
Plant cell staining
Another study used ROS probes to stain stigmas under different conditions i.e. (unpollinated (UP), after pollination, self-incompatibility-pollination (SI), and compatible pollination (CP)), and proved that ROS was involved in self-incompatibility (SI) responses in cruciferous plants.
Results indicated that, compared with the steady-state level of ROS in unpollinated (UP) stigmas, ROS started to show a significant increase as early as 1 min after self-pollination, and reached the maximum level, about 3-fold that of unpollinated stigmas within 30 min (Figure 3A). Co-staining stigmas with H2DCFDA and the cell wall indicator propidium iodide (PI) showed that ROS was located in the cytoplasm of papilla cells, near the periphery of the plasma membrane (Figure 3B). These results suggested that the increase in stigmatic ROS was a specific response induced by SI[2].
Protoplast staining
Furthermore, H2DCFDA can also be used for protoplast staining. As shown in figure 4, H2DCFDA was used to stain protoplast to detect the production of ROS and live-cell imaging was used to track the whole process. The results showed that the ROS levels in protoplasts were continuously increased[3].
Note: The concentration and incubation time of H2DCFDA staining for different samples, like microbes, plant leaves, protoplasts, stigmas, etc., need to be adjusted accordingly.
The use of H2O2, ?O2??and ?OH in vitro
HKPerox-2: As a stable reactive oxygen species, H2O2?also plays an important role in oxidative damage and cell signal transduction. Figure 5A shows HKPerox-2’s selectivity for H2O2?with high fluorescence intensity. When the cells were treated with H2O2, HKPerox-2 reached to the highest fluorescence intensity within 30 minutes as shown in Figure 5B. The fluorescence of HKPerox-2 is shown in Figure 5C which is higher as compared to the control.
HKSOX-1 and HKOH-1r are superoxide anion radical (?O2?) and Hydroxyl radical (?OH) fluorescent probes, which have almost the same use as H2DCFDA and HKPerox-2.
Related products
H2DCFDA: H2DCFDA is used to detect the intracellular reactive oxygen species (ROS).
HKSOX-1: HKSOX-1 has excellent selectivity and sensitivity to superoxide anion radicals. It can be used for imaging and detection of endogenous superoxides in living cells.
HKPerox-2: HKPerox-2 is a green fluorescence probe that has high selectivity for H2O2.
HKOH-1r: HKOH-1r is used to detect endogenous hydroxyl radical ?OH in living cells.
https://www.medchemexpress.com/literature/ros-probes-all-eyes-are-focused-here.html ??