Chel A [6 (link)(7,8-epoxy-styryl)-5-acetoxy-5,6-dihydro-2-pyrone] was isolated from Goniothalamus cheliensis by the Kunming Institute of Botany, Chinese Academy of Sciences, Kunming, China ref. [5 (link)]. Dichlorofluorescein diacetate (DCFH-DA) and hydroethidine (HE) were from purchased from Invitrogen (Carlsbad, CA, USA). Chemicals MG132 and cycloheximide (CHX) were purchased from Calbiochem (San Diego, CA, USA). Dichlorofluorescein diacetate (DCFH-DA) and hydroethidine (HE) were from purchased from Invitrogen (Carlsbad, CA, USA).
Hydroethidine he
Manufactured by Thermo Fisher Scientific
Sourced in United States
Hydroethidine (HE) is a fluorescent dye used as a probe to detect and measure superoxide (O2•-) levels in biological systems. It functions by undergoing oxidation in the presence of superoxide, resulting in the formation of a fluorescent product. HE is commonly used in various research applications to investigate the role of superoxide in cellular processes and signaling pathways.
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Lab products found in correlation
Dichlorofluorescein diacetate dcfhda, by Thermo Fisher Scientific (2 mentions)
Enzychrom nadp nadph assay kit, by BioAssay Systems (2 mentions)
3 4 5 dimethylthiazol 2 yl 2 5 diphenyltetrazolium bromide mtt assay kit, by Merck Group (2 mentions)
Mip 2, by R&D Systems (2 mentions)
Antibodies against erk, by Santa Cruz Biotechnology (2 mentions)
Mcp 1, by R&D Systems (2 mentions)
Interferin sirna transfection reagent, by Polyplus Transfection (2 mentions)
Rabbit ab against c jun n terminal kinases jnk, by Cell Signaling Technology (2 mentions)
Mouse ab against phospho jnk, by BD (2 mentions)
Screen quest fluo 8 medium removal calcium assay kit, by AAT Bioquest (2 mentions)
N acetylcysteine, by Merck Group (2 mentions)
Fura 2 am, by Thermo Fisher Scientific (1 mentions)
Pluronic f68, by Merck Group (1 mentions)
Cetyl palmitate, by Thermo Fisher Scientific (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Cytochrome c, by Merck Group (1 mentions)
Ficoll paque plus, by GE Healthcare (1 mentions)
α tocopherol, by Merck Group (1 mentions)
Xanthine oxidase, by Merck Group (1 mentions)
19 protocols using hydroethidine he
1
Isolation and Characterization of Chel A
2
Lipid-based Nanoparticle Preparation Protocol
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Soybean oil, Pluronic® F68, CTAB, PLGA (50:50, molecular weight 30–60 kDa), and poly(vinyl alcohol) were provided by Sigma-Aldrich Co. (St Louis, MO, USA). Cetyl palmitate was purchased from Acros (Geel, Belgium). SME was supplied by Croda (East Yorkshire, UK). Soybean phosphatidylcholine (SPC) (Phospholipon® 80H) was from American Lecithin (Oxford, CT, USA). Hydroethidine (HE) and Fura-2 AM were purchased from Thermo Fisher Scientific (Waltham, MA, USA). 1,2-Bis(2-aminophenoxy) ethane-N,N,N’,N’-tetraacetic acid (BAPTA-AM) was from Calbiochem (Billerica, MA, USA). Rhodamine 800 was provided by AAT Bioquest (Sunnyvale, CA, USA).
3
Neutrophil Signaling Pathway Evaluation
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Cytochalasin B (CB), cytochrome c, dextran, triton-X-100, DMSO, fMLF, 1,1-diphenyl-2-picryl-hydrazyl (DPPH), xanthine oxidase, and α-tocopherol were obtained from MilliporeSigma (St. Louis, MO, USA). Hydroethidine (HE) and Hank’s balanced salts solution (HBSS) were acquired from Thermo Fisher Scientific Inc. (Waltham, MA, USA). Biological Industries (Beth Haemek, Israel) was the source of trypan blue. N-Methoxysuccinyl-Ala-Ala-Pro-Val p-nitroanilide was obtained from Calbiochem Research Biochemicals (La Jolla, CA, USA). We purchased 2-(4-Iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium monosodium salt (WST-1) from Dojindo Molecular Technologies, Inc. (Kumamoto, Japan). Ficoll-paque plus was procured from GE Healthcare Systems (Little Chalfont, Buckinghamshire, UK). The antibodies against protein kinase B (Akt), phospho-Akt (Ser473), phospho-p38, p38 mitogen-activated protein kinase (MAPK), c-Jun N-terminal kinase (JNK), phospho-JNK, extracellular signal-regulated kinase (ERK), and phospho-ERK were acquired from Cell Signaling Technology (Beverly, MA, USA). The fluo-3-acetomethoxyester was acquired from Molecular Probes biotechnology company (Eugene, OR, USA). Anti-CD11b antibodies were bought from Abcam (Abcam, Cambridge, MA, USA).
4
Preparation and Purification of Hydroethidine Oxidation Products
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Hydroethidine (HE) was purchased from Invitrogen (Carlsbad, CA). A stock solution of HE (20 mM) was prepared in deoxygenated DMSO and stored at −80 °C until use. Ethidium cation (bromide salt) was purchased from Sigma-Aldrich (St. Louis, MO). The hydroxylated oxidation product from HE (2-hydroxyethidium, 2-OH-E+) was prepared by reacting the probe with Fremy’s salt45 (link),57 (link). The dimeric product (E+–E+) was prepared by reacting the probe with excess potassium ferricyanide57 (link). Synthesized standards of all oxidation products of HE were purified by high-performance liquid chromatography (HPLC).
5
Detection of Superoxide and Hydroxyl Radicals
6
Cytometric Determination of Parasite PPE
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The PPE of parasite cultures was determined by flow cytometry, as previously described [26 (link), 27 (link)]. Briefly, 5 µl of the cultures was collected from the bottom of the wells and centrifuged at 450 g for 1 min at 4 ℃. The supernatant was discarded and the cell pelle washed twice with 150 μl of phosphate buffer saline (PBS) pH 7.2, following which the cell pellet was suspended in 200 μl of 25 μg/μl hydroethidine (HE) (Invitrogen, Carlsbad, CA, USA), incubated in 5% CO2 in an incubator at 37 ℃ for 20 min in the dark and ten washed twice with 200 μl of PBS to remove the excess HE. The supernatant was then discarded, and the cell pellet was suspended in 200 μl of fresh PBS. The suspended cells were analyzed by flow cytometry using a Guava® easyCyte flow cytometer (Luminex Corp., Austin, TX, USA) at a ratio of 800–1000 cells/µl with 20,000 events collected. The results were analyzed by FCS Express v6 (De Novo Software, Glendale, CA, USA). Normal, uninfected horse and cattle RBC were used as a negative control for the flow cytometric analysis.
7
Flow Cytometric Assay for Erythrocyte Infection
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Flow cytometric assay was performed as described by Wyatt et al. (1991) . Briefly, cultures were centrifuged at 450 × g for 5 min at 4 °C. The supernatant was discarded, the cell pellet was washed twice with 150 μl of phosphate buffer saline (PBS) pH 7.2 and then, the cell pellet was resuspended in 200 μl of 25 μg/μl Hydroethidine (HE) (Invitrogen) and incubated in 5% CO2 incubator at 37 °C for 20 min in the dark. After incubation, excess of HE was washed from the cells by the addition of 200 μl of PBS followed by centrifugation at 450 × g for 5 min at 4 °C. The supernatant was discarded and the cell pellet was resuspended in 200 μl of fresh PBS. Then, resuspended cells were transferred to samples tube containing approximately 3 ml of PBS containing 0.2% sodium azide for analysis by flow cytometry using a FACSCaliber (Becton Dickinson) at a flow rate of approximately 2500 events/s with 50,000 events collected. Data was analyzed by FCS Express 3 software (De Novo Software). Stained non-infected erythrocytes were used as a negative control and gated to determine the percentage between infected and non-infected erythrocytes.
8
Multiparametric Sperm Oxidative Stress Assay
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Hydroethidine (HE; Invitrogen, Thermo Fisher Scientific, Eugene, OR, USA) and 20, 70 -dichlorodihydrofluorescein diacetate (DCFDA; Invitrogen, Thermo Fisher Scientific, Eugene, OR, USA) were used to detect superoxide (O2−) and hydrogen peroxide (H2O2), respectively, while Hoechst 33258 (HO) was added to permit the simultaneous differentiation of living and dead cells. Aliquots (300 μL) of semen extended to a concentration of approximately 2 × 106 spermatozoa/mL (spz/mL) with Buffer B were stained using each of the following: 3 µL of HO (40 mM), 3 µL HE (40 mM), and 3 µL DCFDA, (2 mM). The samples were gently mixed and incubated at 38 °C for 30 min before analysis. Using dot-plots for HO/HE and HO/DCFDA, the following populations were quantified: ROS Live SO−; ROS Live SO+; ROS Dead SO+; ROS Dead H2O2−; ROS Dead H2O2+; ROS Live H2O2−; and ROS Live H2O2+.
9
ROS Production Measurement in HaCaT Cells
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The cellular ROS production was measured by treating HaCaT cells with 10 μM hydroethidine (HE; Molecular Probes, Eugene, OR) for 30 min at 37 °C. Fluorescence-stained cells were then analyzed with BD FACS Aria III (BD Biosciences). For measurement of cellular ROS production, HaCaT cells were treated with diluted LP (1/2, 1/4) for 24 h and then were treated with 10 μM of 5-(and 6)-carboxyl-2′,7′-dichlorodihydro fluorescein diacetate (DCF-DA; Molecular Probes) for 30 min at 37 °C. Intracellular ROS levels was detected using a FACScan flow cytometer (BD Biosciences) with excitation and emission settings at 488 and 530 nm, respectively.
10
Intracellular ROS Evaluation in U937 Cells
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Intracellular ROS generation in U937 cells was evaluated by flow cytometry using the fluorescence generated in the cells loaded with the sensitive fluorescent probes, a H2O2 sensitive dye, dichlorofluorescein diacetate (DCFH‐DA) (Molecular probes, Eugene, OR, USA). Hydroethidine (HE) (Molecular Probes) was used to determine superoxide generation O2−. The cells were pre‐incubated with Pt‐NPs at a dose of 300 μM for 3 and 6 hrs then exposed to He‐CAP for 4 min., immediately after post‐treatment; DCFH‐DA was added at a final concentration of 10 μM and HE was added at a final concentration of 5 μM. The fraction of fluorescence positive cells was measured by flow cytometry as the proportion of cells containing intracellular ROS.
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