superoxide indicator; blue-fluorescence dye

General procedure:
Preparation of dihydroethidium working solution
1.1 Prepare the stock solution by dissolving 1 mg of dihydroethidium in 0.31 mL DMSO to make 10 mM stock solution.
Note: Avoid repeated freezing and thawing cycle, it is advised that the stock solution be stored at -20°C or -80°C and away from light.
1.2 Preparation of dihydroethidium working solution: Dilute the stock solution into 1–10 μM working solution in PBS or serum-free cell culture medium.
Note: You may adjust the concentration of Dihydroethidium working solution based on your specific needs.
Cell staining
2.1: Suspension cells: Centrifuge 1000g of suspended cells for 3–5 minutes at 4°C; discard supernatant. Use PBS to wash twice, for five minutes each time.
Adherent cells: To isolate the cells and create a single cell suspension, discard the cell culture medium and add trypsin. Discard the supernatant after centrifuging at 1000g for three to five minutes at 4°C. Use PBS to wash twice, for five minutes each time.
2.2 Add 1 mL of the Dihydroethidium working solution, then let it sit at room temperature for half an hour.
2.3 Discard the supernatant after centrifuging at 400 g for three to four minutes at 4°C.
2.4 Wash the cells with PBS twice, five minutes/each time.
2.5 Re-suspend the cells in PBS or serum-free cell culture medium, then use a flow cytometer or fluorescence microscope to observe them. Storage for a year at -20°C with protection from light.
Precautions:
1. The original solution should be kept refrigerated at -20°C or -80°C, shielded from light, and avoid freezing and thaw too frequently.
2. You may adjust the concentration of Dihydroethidium working solution based on your specific needs.
3. This product is intended for research use only.
4. Please wear a lab coat and disposable gloves for your own health and safety when operating.

Oxidative stress-related indicators in OXA-induced HSOS in mice[2]
The microarray results suggested that oxidative stress may serve an important role in OXA-induced HSOS. Therefore, the levels of common oxidative stress markers were determined and changes in ROS were observed by DHE staining. MDA is a lipid peroxidation product in vivo, which can cause cytotoxicity. The MDA levels in the mice livers were significantly increased following the administration of 10 mg/kg OXA (P<0.05; Fig. 7A). SOD is an antioxidant metal enzyme that can catalyze the superoxide anion radical and provide cellular defenses against ROS. CAT is also an antioxidant enzyme that protects cells from the toxicity of H2O2. GSH has antioxidant and integrative detoxification effects. OXA markedly reduced the levels of SOD, CAT and GSH in the mice livers (P<0.05; Fig. 7B-D). Furthermore, DHE probe technology was used to analyze changes in ROS levels. The results showed that the ROS levels in the livers of the mice in the OXA group were increased in a dose-dependent manner (Fig. 7E). Taken together, these data confirmed that oxidative stress may have an important role in the liver damage of mice with OXA-induced HSOS.

Hydroethidine (HE) or dihydroethidium and its mitochondria-targeted analog conjugated to a triphenylphosphonium moiety (MitoSOXTM Red) react rapidly with superoxide (k ∼106 m−1 s−1), forming a specific hydroxylated marker product, 2-hydroxyethidium (2-OH-E+) or 2-hydroxymitoethidium (2-OH-Mito-E+)[1].

Intracellular ROS measurements[3]
Two dyes were used to detect ROS: 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate acetyl ester (CM-H2DCFDA: excitation wavelength, 504 nm; emission wavelength, 524 nm) and dihydroethidium (DHE; excitation wavelength, 518 nm; emission wavelength, 605 nm). Both dyes were reconstituted with DMSO. Monocytes and THP-1 cells were incubated with dye at 37°C for 30 min. Cell pellets were resuspended in media and incubated for 15 min at 37°C to allow esterases to cleave CM-H2DCFDA to trap it inside the cell. Cells were then incubated with a2NTD, PMA, or media alone with or without 100 μM diethyldithiocarbamic acid sodium salt trihydrate. Two methods were used to detect intracellular ROS. After a2NTD stimulation, cells were analyzed for fluorescence via flow cytometer on an LSR II or via a microplate reader in a 96-well black plate

Staining of dihydroethidium (DHE)-reactive oxygen species (ROS)[2]
Frozen liver sections (−26°C; 6 µm thickness) were warmed at room temperature and mounted with an anti-fluorescence quenching solution for 5 min. ROS dye solution was added dropwise and sections were incubated for 30 min at 37°C in the dark. Subsequently, the sections were washed three times with phosphate-buffered saline (PBS, pH 7.4) and DAPI staining solution was added dropwise and incubated for 10 min at room temperature in the dark. After washing three times with PBS and drying, the sections were mounted with an anti-fluorescence quenching solution. The sections were observed under a fluorescence microscope and images were captured.

[1]. Zielonka J, et al. Global profiling of reactive oxygen and nitrogen species in biological systems: high-throughput real-time analyses. J Biol Chem. 2012 Jan 27;287(5):2984-95.
Model establishment and microarray analysis of mice with oxaliplatin‑induced hepatic sinusoidal obstruction syndrome. Mol Med Rep . 2022 Nov;26(5):346.
[3]. Tumor-associated a2 vacuolar ATPase acts as a key mediator of cancer-related inflammation by inducing pro-tumorigenic properties in monocytes. J Immunol. 2011 Feb 1;186(3):1781-9.

Cancer-related inflammation profoundly affects tumor progression. Tumor-associated macrophages (TAMs) are known regulators of that inflammation, but the factors that initiate cancer-related inflammation are poorly understood. Tumor invasiveness and poor clinical outcome are linked to increased expression of cell surface-associated vacuolar adenosine triphosphatases. The a2 isoform vacuolar adenosine triphosphatase is found on the surface on many solid tumors, and we have identified a peptide cleaved from a2 isoform vacuolar adenosine triphosphatase called a2NTD. a2NTD has properties necessary to induce monocytes into a pro-oncogenic TAM phenotype. The peptide upregulated both pro- and anti-inflammatory mediators. These included IL-1β and IL-10, which are important in promoting inflammation and immune escape by tumor cells. The secretion of inflammatory cytokine IL-1β was dependent on ATP, K(+) efflux, and reactive oxygen species, all mediators that activate the inflammasome. These findings describe a mechanism by which tumor cells affect the maturation of TAMs via a nontraditional cytokine-like signal, the a2NTD peptide.[3]

---

Herein we describe a high-throughput fluorescence and HPLC-based methodology for global profiling of reactive oxygen and nitrogen species (ROS/RNS) in biological systems. The combined use of HPLC and fluorescence detection is key to successful implementation and validation of this methodology. Included here are methods to specifically detect and quantitate the products formed from interaction between the ROS/RNS species and the fluorogenic probes, as follows: superoxide using hydroethidine, peroxynitrite using boronate-based probes, nitric oxide-derived nitrosating species with 4,5-diaminofluorescein, and hydrogen peroxide and other oxidants using 10-acetyl-3,7-dihydroxyphenoxazine (Amplex® Red) with and without horseradish peroxidase, respectively. In this study, we demonstrate real-time monitoring of ROS/RNS in activated macrophages using high-throughput fluorescence and HPLC methods. This global profiling approach, simultaneous detection of multiple ROS/RNS products of fluorescent probes, developed in this study will be useful in unraveling the complex role of ROS/RNS in redox regulation, cell signaling, and cellular oxidative processes and in high-throughput screening of anti-inflammatory antioxidants.[2]

---

Hepatic sinusoidal obstruction syndrome (HSOS) is a serious side effect of oxaliplatin (OXA) treatment. The present study aimed to establish a reproducible mouse model of OXA‑induced HSOS and to preliminarily explore the underlying molecular mechanisms using mRNA microarray analysis. A total of 45 C57BL/6 male mice were randomly divided into five groups: Control, 5 mg/kg OXA, 10 mg/kg OXA, 15 mg/kg OXA and 20 mg/kg OXA. The mice were respectively injected intraperitoneally with 5% glucose solution, or 5, 10, 15 or 20 mg/kg OXA solution once a week for 6 consecutive weeks. The body weight of the mice was recorded every day. The serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were determined. Hematoxylin and eosin staining, Sirius red staining and scanning electron microscopy were used to identify pathological changes. mRNA microarray was used to analyze changes in the gene expression profiles mainly from the functional aspects of Gene Ontology and the Kyoto Encyclopedia of Genes and Genomes. The oxidation mechanism was verified by measuring oxidative stress‑related markers and reactive oxygen species with dihydroethidium probe technology, according to the microarray results. Among all of the OXA groups, 10 mg/kg OXA resulted in an acceptable survival rate of 78%. The mice showed obvious splenomegaly, increases in serum levels of ALT and AST, aggravation of liver pathological injuries and hepatic sinusoidal injuries. The microarray results suggested that mRNA expression changes after OXA treatment were associated with 'oxidative stress', 'coagulation function', 'steroid anabolism' and 'pro‑inflammatory responses'. The results confirmed that OXA aggravated oxidative damage in the livers of the mice. The present study successfully established a mouse model of OXA‑induced HSOS and preliminarily analyzed the underlying molecular mechanisms involved, thus laying a foundation for a subsequent in‑depth study.[1]

C21H21N3

315.41

315.17

C, 79.97; H, 6.71; N, 13.32

104821-25-2

128682

Pale purple to light pink solid powder

3.06

55.92

NC1=CC=C2C3=C(C=C(N)C=C3)C(C4=CC=CC=C4)N(CC)C2=C1

XYJODUBPWNZLML-UHFFFAOYSA-N

InChI=1S/C21H21N3/c1-2-24-20-13-16(23)9-11-18(20)17-10-8-15(22)12-19(17)21(24)14-6-4-3-5-7-14/h3-13,21H,2,22-23H2,1H3

5-ethyl-6-phenyl-6H-phenanthridine-3,8-diamine

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: ≥ 2.5 mg/mL (7.93 mM) (saturation unknown) in 10% DMSO + 40% PEG300 +5% Tween-80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 + to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

---

 (Please use freshly prepared in vivo formulations for optimal results.)