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Hydroethidine

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Hydroethidine is a fluorescent dye used in cell biology research. It is a redox-sensitive probe that can be used to detect the presence of superoxide radicals in cells. The core function of Hydroethidine is to provide a fluorescent readout for the detection of superoxide levels in biological samples.

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Lab products found in correlation

Dapi solution, by Vector Laboratories (2 mentions) Facscalibur, by BD (1 mentions) Lysis buffer, by BD (1 mentions) Sliding microtome, by Thermo Fisher Scientific (1 mentions) Confocal microscopy, by Zeiss (1 mentions) Eclipse ti2 series, by Nikon (1 mentions) Sodium phosphate dibasic heptahydrate, by Merck Group (1 mentions) Sodium phosphate monobasic anhydrous, by Merck Group (1 mentions) Imidazole, by Merck Group (1 mentions) Superoxide dismutase bovine, by Merck Group (1 mentions) Sodium azide, by Merck Group (1 mentions) 2 5 dihydroxybenzoic acid dhb, by Merck Group (1 mentions) Ethidium bromide, by Merck Group (1 mentions) Ultrapure dnase rnase free distilled water, by Bioneer (1 mentions) Glucose, by Merck Group (1 mentions) Accu chek, by Roche (1 mentions) I taqtm dna polymerase, by iNtRON Biotechnology (1 mentions) Penicillin, by Thermo Fisher Scientific (1 mentions) Streptomycin, by Thermo Fisher Scientific (1 mentions) Hepes, by Thermo Fisher Scientific (1 mentions)

22 protocols using hydroethidine

1

Intracellular ROS Quantification in HDFs

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The intracellular concentration of reactive oxygen species of HDFs was measured by using an oxidation-sensitive fluorescent probe dye, 2′,7′-dichlorodihydrofluorescein diacetate (DCF-DA) and hydroethidine (Sigma Co.) [15 (link)]. To measure intracellular ROS, the cells were incubated for 1 hr at 37°C with HBSS containing 33 μM DCF-DA (Molecular Probes) or 1 μM hydroethidine (Sigma Co.). The samples were then immediately observed under confocal fluorescence microscope (Olympus, Japan). The images were obtained by overlaying fluorescent images to differential interference contrast images. Also, DCF fluorescence was detected by FACStar flow cytometer (Becton Dickinson). For each sample, 10,000 events were collected. Reactive oxygen species production was expressed as mean fluorescence intensity (MFI), which was calculated by CellQuest software. Additionally, the cells were incubated for 1 hr at 37°C with HBSS containing 33 mM DCF-DA (Invitrogen Molecular Probes, Eugene, OR). The samples were then immediately observed under fluorescence microscopy.
Noh E.M., Park J., Song H.R., Kim J.M., Lee M., Song H.K., Hong O.Y., Whang P.H., Han M.K., Kwon K.B., Kim J.S, & Lee Y.R. (2016). Skin Aging-Dependent Activation of the PI3K Signaling Pathway via Downregulation of PTEN Increases Intracellular ROS in Human Dermal Fibroblasts. Oxidative Medicine and Cellular Longevity, 2016, 6354261.
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2

Neutrophil Phagocytosis and Respiratory Burst Assay

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The flow cytometry was used for the analysis of phagocytosis and respiratory burst of neutrophils as described previously 11. Neutrophils from whole blood samples were adjusted to a cell density of 3 × 106 cells·mL−1, followed by in vitro coculture with fluorescein isothiocyanate (FITC)‐labeled bacteria [heat‐killed Staphylococcus aureus (HKSA)] at multiplicity of infection of 10 : 1 for 60 min or with PMA (800 ng·mL−1) for 15 min at 37 °C. This was followed by the addition of 25 mL of a 2800 ng·mL−1 solution of hydroethidine (Sigma, San Francisco, CA, USA) and incubation for 5 min at 37 °C. Erythrocytes were excluded with lysis buffer (BD Biosciences, San Jose, CA, USA) before analysis by flow cytometry (FACSCalibur; BD Biosciences). The number of neutrophils with phagocytic function in 1‐mL samples of whole blood was calculated by multiplying absolute neutrophil counts (determined using automated blood counter) with the percentage of phagocytosis (analyzed by flow cytometry) and dividing the obtained value by 100. Respiratory burst function, which occurs after phagocytosis, was subsequently calculated by multiplying the number of phagocytosing neutrophils (calculated earlier) by the percentage of respiratory burst (analyzed by flow cytometry) and dividing the obtained value by 100.
Yu Q., Li Y., Wang H, & Xiong H. (2019). TSLP induces a proinflammatory phenotype in circulating innate cells and predicts prognosis in sepsis patients. FEBS Open Bio, 9(12), 2137-2148.
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3

Evaluating ROS Production in Skin

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ROS production in tail skin was evaluated ex vivo using hydroethidine (Sigma-Aldrich), which reacts with ROS to produce fluorescent dye hET. Samples were incubated at 37 ºC for 3 h in a solution with 5 mM EDTA in PBS, containing 2 mM mALA in the case of treated samples. hydroethidine was added to a final concentration of 3.2 μM. After 1 h, the skin was irradiated with 10 J/cm2 of 636 nm light and fixed.
For in vivo detection of ROS produced in back skin during photodynamic treatment, the ROS sensitive DHF-DA (Sigma-Aldrich) was used. In order to assess the inhibitory effect of AA, two independent groups of animals were established for each time point for normalizing the obtained signal (mALA+Light/Light) with respect to AA-animals. After dorsal skin shaving and PT treatment application as previously described (Metvix® 2.5 h in dark followed by the irradiation with 2.5 J/cm2 of 636 nm light), 1 mg/ml DHF-DA in 50% ethanol was topically applied on the tissue at different time points after the irradiation (5, 20 and 100 min). ROS levels generated in the skin were determined 45 min after the application of DHF-DA in all cases, using an IVIS® Lumina 2 imaging system (Xenogen), by measuring the fluorescent signal emitted by the fluorescein produced through the oxidation of DHF-DA. The filters setting were 445–490 nm for the excitation and 515–575 nm for the emission.
Carrasco E., Calvo M.I., Blázquez-Castro A., Vecchio D., Zamarrón A., de Almeida I.J., Stockert J.C., Hamblin M.R., Juarranz Á, & Espada J. (2015). Photoactivation of ROS production in situ transiently activates cell proliferation in mouse skin and in the hair follicle stem cell niche promoting hair growth and wound healing. The Journal of investigative dermatology, 135(11), 2611-2622.
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4

In Situ Visualization of Oxidants

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hydroethidine histochemistry was performed for in situ visualization of the O2- and O2-derived oxidants. Three days after pKr-2 injection, hydroethidine (1 mg/kg in PBS containing 1% dimethylsulfoxide; Sigma, St. Louis, MO, USA) was intravenously administered through tail vein. After 45 min from hydroethidine injection, the brain tissues were prepared, as previously described [40 (link),52 (link)]. The brain tissues were cut into 40 µm using a sliding microtome (Thermo Scientific, Walldorf, Baden-Württemberg, Germany) and the tissues were mounted on gelatin-coated slides. The oxidized hydroethidine product, ethidium, examined by confocal microscopy (Carl Zeiss), and then merged with DAPI solution (Vector Laboratories). Image J quantified the obtained images in each group (National Institutes of Health, USA).
Jeong J.Y., Chung Y.C, & Jin B.K. (2019). Interleukin-4 and Interleukin-13 Exacerbate Neurotoxicity of Prothrombin Kringle-2 in Cortex In Vivo via Oxidative Stress. International Journal of Molecular Sciences, 20(8), 1927.
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5

Superoxide Anion Detection in Liver

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A superoxide anion (O2•−) was detected using chemiluminescence. Liver O2•− production was evaluated in the CTRL and IUGR males at birth and at 6 months of age as previously described [30 (link),41 (link)]. Deparaffinized liver sections (5-μm thick) were stained with hydroethidine (2 μM, Sigma–Aldrich) and incubated in a light-protected humidified chamber at 37 °C for 30 min. The sections were rinsed and mounted using Fluoromount g mounting medium with 4′6-diamidino-2-phenylindole (DAPI; Life Technologies Europe B.V, Zug, Switzerland). A negative control was established through incubation without hydroethidine. Images were obtained blindly using an inverted fluorescent microscope (Eclipse Ti2 Series-Nikon) by a single examiner (C.Y.). Fluorescence was evaluated with ImageJ software, and liver autofluorescence was subtracted.
Keshavjee B., Lambelet V., Coppola H., Viertl D., Prior J.O., Kappeler L., Armengaud J.B., Chouraqui J.P., Chehade H., Vanderriele P.E., Allouche M., Balsiger A., Sarre A., Peyter A.C., Simeoni U, & Yzydorczyk C. (2022). Stress-Induced Premature Senescence Related to Oxidative Stress in the Developmental Programming of Nonalcoholic Fatty Liver Disease in a Rat Model of Intrauterine Growth Restriction. Antioxidants, 11(9), 1695.
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6

Synthesis and Characterization of HMBR

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4-hydroxy-3-methylbenzylidene-rhodanine (HMBR) was synthesized as previously described29 (link). Sodium phosphate dibasic heptahydrate (≥98%), sodium phosphate monobasic anhydrous (≥96%), Imidazole (≥99%), Hydroethidine (HE) (≥95%), Catalase from bovine liver (≥70%, ≥10kU/mg), Superoxide Dismutase bovine (SOD) (≥90%, ≥2.5kU/mg), D2O (99.9%) and sodium azide (99.5%) were obtained from Sigma-Aldrich and used as received.
Pimenta F.M., Chiappetta G., Le Saux T., Vinh J., Jullien L, & Gautier A. (2017). Chromophore Renewal and Fluorogen-Binding Tags: A Match Made to Last. Scientific Reports, 7, 12316.
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7

Glucose Detection via CeO2 Nanoparticles

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All DNA oligonucleotides used in this study were synthesized and purified with high performance liquid chromatography (HPLC) by Bioneer® (Daejeon, Korea). The sequence information of oligonucleotides employed in this study is in Table S1. The personal glucose meter (PGM) whose dynamic range for glucose detection is from 0.6 to 33 mM was purchased from Accu-Chek (Roche, Basel, Switzerland). Cerium (IV) oxide nanoparticle (CeO2 NP), sodium acetate, glucose, ethidium bromide (EtBr), hydroethidine, and 2,5-dihydroxybenzoic acid (DHB) were purchased from Sigma-Aldrich (St. Louis, MO, USA). The deoxynucleoside triphosphate (dNTP) and i-TaqTM DNA polymerase were purchased from Intron Biotechnology Inc. (Daejeon, Korea). Ultrapure DNase/RNase-free distilled water (DW) purchased from Bioneer® was used in all experiments. All chemicals used in this study were of analytical grade.
Kim H.Y., Park K.S, & Park H.G. (2020). Glucose oxidase-like activity of cerium oxide nanoparticles: use for personal glucose meter-based label-free target DNA detection. Theranostics, 10(10), 4507-4514.
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8

Neutrophil Activation Assay Protocol

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RPMI-1640 medium, HEPES, penicillin, and streptomycin were purchased from Invitrogen (Carlsbad, CA, USA). Hydroethidine, Histopaque 1077, PMA (phorbol myristate acetate), LPS (lipopolysaccharide), Triton X-100, and propidium iodide were supplied by Sigma Chemical Co. (St. Louis, MO). Hydroethidine and PMA were dissolved in DMSO (dimethyl sulfoxide). The final concentration of DMSO in the assay medium did not exceed 0.01%. A preliminary experiment showed that DMSO at this concentration is not toxic for neutrophils and does not interfere in the results obtained [15 (link)]. Reagents, water, and plastic wares used in the experiments were all endotoxin-free.
Borges L.D., Bortolon J.R., Santos V.C., de Moura N.R., Dermargos A., Cury-Boaventura M.F., Gorjão R., Pithon-Curi T.C, & Hatanaka E. (2014). Chronic Inflammation and Neutrophil Activation as Possible Causes of Joint Diseases in Ballet Dancers. Mediators of Inflammation, 2014, 846021.
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9

Invasion Inhibition Antibody Assay Protocol

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The method to study invasion inhibitory antibodies has been described previously [68 (link)]. Two Ugandan P. falciparum isolates [64 (link)], UAM37 (from a patient with mild malaria, containing both FC27 and 3D7 alleles of MSP2), and UAS31 (from a patients with severe malaria, containing the 3D7 allele of MSP2) were cultured in vitro (for a couple of months after collection from the patients) in AB+ non-immune Swedish serum and gassed with 90 % NO2, 5 % O2 and 5 % CO2 and placed in a shaker incubator. In brief, parasites were synchronized (5 % sorbitol, v/w) before assay start, and at the day of the assay the majority of the parasites were at late-pigmented trophozoite stage. 50 μl of parasite suspensions were cultured for one cycle in 96 well plates. 5 μl of dialyzed test plasma was added to each well and all samples were run in duplicate. Plates were incubated in a sealed, humidified, gassed box and put in an incubator for 48 hours at 37 °C. Parasitemia was estimated using hydroethidine (10 ug/ml; Sigma Aldrich) in a flow cytometer (FACS Scan; BD) after approximately 48 hours (determined by the parasite stage). Parasite invasion for each sample was measured in comparison to controls (invasion in presence of dialyzed non-immune plasma).
Reddy S.B., Anders R.F., Cross N., Mueller I., Senn N., Stanisic D.I., Siba P.M., Wahlgren M., Kironde F., Beeson J.G, & Persson K.E. (2015). Differences in affinity of monoclonal and naturally acquired polyclonal antibodies against Plasmodium falciparum merozoite antigens. BMC Microbiology, 15, 133.
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10

Measurement of Reactive Oxygen Species

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The thalli (0.1 g fresh weight) were ground into a powder form in liquid nitrogen, and 3 ml of 0.05 M phosphate buffer solution (PBS) (pH = 7.4) was added. This was then centrifuged at 12,000 rpm 4°C for 20 min, and the supernatant was carefully removed to measure the ROS content ( O2∙- , H2O2, ∙OH ). The supernatant was incubated in 100 µM of hydroethidine (Sigma-Aldrich Co., USA) for 1 h (37°C) without light. The corresponding excitation wavelength was 480 nm, and the emission wavelength was 590 nm to determine the O2∙- [85 (link)]. The supernatant was incubated in 15 µM DCFH-DA (Solarbio Science & Technology Co., Ltd., Beijing) for 45 min (37°C) and protected from light. To determine the H2O2, the corresponding excitation wavelength was 488 nm and the emission wavelength was 530 nm [86 (link)]. The supernatant was incubated in 240 µM of 1,3-cyclohexanedione (CHD) (Sigma-Aldrich Co., USA), 420 mM pH 3.6 NH4Ac, and 105 µM of dimethyl sulfoxide (DMSO) under dark conditions for 20 min (95°C). To determine the ∙OH , the corresponding excitation wavelength was 400.5 nm and the emission wavelength was 452.3 nm [87 (link)].
Xu J., Zhao X., Zhong Y., Qu T., Sun B., Zhang H., Hou C., Zhang Z., Tang X, & Wang Y. (2024). Acclimation of intertidal macroalgae Ulva prolifera to UVB radiation: the important role of alternative oxidase. BMC Plant Biology, 24, 143.
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