Diphenyleneiodonium chloride dpi

Manufactured by Merck Group

Sourced in United States, Germany

Diphenyleneiodonium chloride (DPI) is a chemical compound used as a laboratory tool. It functions as an inhibitor of the enzyme NADPH oxidase, which is involved in the production of reactive oxygen species. DPI can be used in scientific research to study the role of NADPH oxidase and oxidative stress in various biological systems.

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Diphenyleneiodonium (DPI) (67 citations) Diphenyleneiodonium (33 citations) Diphenyleneiodonium chloride (25 citations) DPI chloride (2 citations)

47 protocols using diphenyleneiodonium chloride dpi

Polarization of Bone Marrow-Derived Macrophages

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BMMФ were stimulated either with IL-4 (10 ng/ml, AF-214-14, Peprotech), lipopolysaccharide (100 ng/ml, Sigma) or dexamethasone (100nM, D4902, Sigma) to respectively achieve M(IL-4), M(LPS) or M(GC) polarization state. In some experiments, BMMФ were treated with C75 (Sigma), Cerulenin (Sigma), Etomoxir (Sigma), pan-AKT inhibitor MK-2206 (1 mM, Cayman Chemical), 25-hydroxycholesterol (10 μM, Cayman chemical), N-acetyl cysteine (10mM, Sigma), Diphenyleneiodonium chloride (DPI, 5 μM, Sigma), L-NG-Nitroarginine methyl ester (L-NAME, 1mM, Cayman Chemical), Allopurinol (100 μM, Sigma), hydrogen peroxide (Sigma), SIRT1 activator II (Sigma), EX-527 (Sigma), Compound C (Sigma), AICAR (Abcam), HMGCoA (Sigma), water-soluble cholesterol (Cholesterol–methyl-β-cyclodextrin, Sigma) or Simvastatin (Sigma).
For fatty acid treatment, palmitic or oleic acid (Cayman) were solubilized at 100mM in absolute ethanol at 60°C. Fatty acid were conjugated at the desired concentration using a sonicator water bath into the macrophage culture medium to avoid endotoxin contamination from BSA^{54 (link)}.

Bidault G., Virtue S., Petkevicius K., Jolin H.E., Dugourd A., Guénantin A.C., Leggat J., Mahler-Araujo B., Lam B.Y., Ma M.K., Dale M., Carobbio S., Kaser A., Fallon P.G., Saez-Rodriguez J., McKenzie A.N, & Vidal-Puig A. (2021). SREBP1-induced fatty acid synthesis depletes macrophages antioxidant defences to promote their alternative activation. Nature metabolism, 3(9), 1150-1162.

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Murine Pancreatic Islet Endothelial Cell Culture

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The murine MS1 (Mile Sven 1) pancreatic islet endothelial cell line, obtained from American Type Culture Collection (ATCC®, CRL-2279^TM), is a commonly used model of microvascular endothelium. MS1 cells were cultured in high glucose (4.5 g/l) Dulbecco’s modified Eagle’s medium (DMEM), supplemented with 5% decomplemented (heat inactivated) fetal bovine serum (FBS), 120 U/mL streptomycin/penicillin and 4 mM L-Glutamine, in 5% CO₂ at 37 °C. MOVAS (ATCC®, CRL-2797^TM) is an established VSMC line from C57BL/6 mice. Cells were grown in DMEM supplemented with 10% FBS, 2 mM L-Glutamine and 0.2 mg/ml G-418 (all reagents obtained from Lonza). For experiments, cells at 80% confluence were growth-arrested by serum starvation for 24 h. Endotoxin-free CsA (Calbiochem, Merck Chemicals) and tacrolimus (USBiological) stock solutions (10 mg/ml) were dissolved in ethanol. CLI-095 (InvivoGen) was used according to manufacturer’s time and dose recommendations. The NF-κB inhibitor parthenolide, the TRIF inhibitor resveratrol and the antioxidants 4′-hydroxy-3′methoxyacetophenone (apocynin) and diphenyleneiodonium chloride (DPI) were from Sigma-Aldrich. These reagents were used at concentration derived from prior dose-response studies in our laboratory or from bibliographic data.

Rodrigues-Diez R., González-Guerrero C., Ocaña-Salceda C., Rodrigues-Diez R.R., Egido J., Ortiz A., Ruiz-Ortega M, & Ramos A.M. (2016). Calcineurin inhibitors cyclosporine A and tacrolimus induce vascular inflammation and endothelial activation through TLR4 signaling. Scientific Reports, 6, 27915.

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Lipid Accumulation and Inflammation in Macrophages

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For Oil Red O (A12989.14 Thermo Fisher Scientific) staining, CD11c⁺ or CD11c⁻ macrophages were fixed in 4% paraformaldehyde for 15 min, washed twice with dH₂O, permeabilized with 60% isopropanol (190764, Sigma for 5 min, stained with an Oil Red O (Stock solution 75 mg in 25 mL 100% isopropanol and working solution 3 parts Oil Red O to 2 parts dH₂O 0.2 μm filtered) for 20 min, images were collected using an Echo RVL2-K microscope.
For FA treatment effects, CD11c⁺ or CD11c⁻ macrophages were treated with 200 μM PA, DPA or control BSA in the presence or absence of 1 mM N-acetyl-L-cysteine (NAC, A7250, Sigma-Aldrich) or 5μM Diphenyleneiodonium chloride (DPI, Sigma-Aldrich) for 24 h for gene expression analysis by real-time PCR. For IL-36 intracellular staining, cells were treated with 200 μM PA, DPA or control BSA for 48 h. After surface staining with anti-mouse CD11c and anti-mouse F4/80, cells were permeabilized for intracellular staining of IL-36α (32103-05161, Assaypro) or IL-36γ (PAL621Mu01, Cloud-clone Corp). For measurement of IL-36γ concentration in cultural supernatants, CD11c⁺ macrophages were treated with 200 μM PA, DPA or control BSA for 48 h. Supernatants were collected and detected using mouse Interleukin-36γ ELISA Kit (MBS288173, Mybiosource) according to the manufacturer’s protocol.

Tamaru Y, & Doi R.H. (1999). Three Surface Layer Homology Domains at the N Terminus of the Clostridium cellulovorans Major Cellulosomal Subunit EngE. Journal of Bacteriology, 181(10), 3270-3276.

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Inhibition of Signaling Pathways in Cell Studies

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p38 inhibitor SB203580 (CAT#13067) [45 (link)], JNK inhibitor SP600125 (CAT#10010466) [46 (link)], JAK inhibitor Tofacitinib CP690,550(CAT#11598) [47 ], and ERK inhibitor U-0126(CAT#70970) [48 (link)–50 (link)], were purchased from Cayman Chemical. Diphenyleneiodonium chloride DPI (CAS#4673-26-1) [39 (link)] was purchased from Sigma-Aldrich. All inhibitors were dissolved in DMSO and used at the indicated concentrations.

West C, & Silverman N. (2018). p38b and JAK-STAT signaling protect against Invertebrate iridescent virus 6 infection in Drosophila. PLoS Pathogens, 14(5), e1007020.

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Immune Cell Labeling and Activation Protocol

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Dihydrorhodamine-123 (DHR-123), Hoechst 33342 and Sytox Green nuclear dyes were purchased from Molecular Probes (Eugene, OR) and stored at −20°C. Phorbyl 12-myristate 13-acetate (PMA) and Diphenyleneiodonium chloride (DPI) were purchased from Sigma Aldrich (Milwaukee, WI). PMA was dissolved in DMSO at a stock solution concentration of 1.6mM and stored at −20°C. DPI was dissolved in de-ionized water at a concentration of 10mM, and stored at room temperature. Recombinant human P-selectin (R&D Systems, Minneapolis, MN) was re-suspended in deionized water (100 μg/mL) and stored at 4°C. Phycoerythrin conjugated anti-human CD14 (Clone 61D3) was purchased from eBioScience (San Diego, CA). AlexaFluor 647 conjugated anti-mouse/human CD11b conjugated (Clone M1/7) and AlexaFluor 790 conjugated anti-human CD45 (Clone H130) were purchased from BioLegend (San Diego, CA).

Moussavi-Harami S., Mladinich K., Sackmann E., Shelef M., Starnes T., Guckenberger D., Huttenlocher A, & Beebe D. (2016). Microfluidic Device for Simultaneous Analysis of Neutrophil Extracellular Traps and Production of Reactive Oxygen Species. Integrative biology : quantitative biosciences from nano to macro, 8(2), 243-252.

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Extracellular Flux Assay Reagents

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DMNQ (2,3 dimethoxynapthoquinone) was purchased from Enzo Life Sciences (Farmingdale, NY, USA). RPMI was obtained from Life Technologies (Grand Island, NY, USA). Materials for the extracellular flux assays were from Seahorse Biosciences (North Billerica, MA, USA). BSA, Oligomycin, FCCP (carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone), Antimycin A and diphenyleneiodonium chloride (DPI) were all purchased from Sigma-Aldrich (St. Louis, MO, USA).

Chacko B.K., Zhi D., Darley-Usmar V.M, & Mitchell T. (2015). The Bioenergetic Health Index is a sensitive measure of oxidative stress in human monocytes. Redox Biology, 8, 43-50.

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Breast Cancer Cell Line Maintenance

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Human breast cancer cell lines, MCF7 [ER(+)] and MDA-MB-468 (triple-negative), were obtained commercially from the ATCC. Both cell lines were maintained in Dulbecco's Modified Eagle Medium (DMEM; GIBCO), supplemented with 10% FBS, 1% Glutamax, and 1% Penicillin-Streptomycin. All cell lines were maintained at 37°C in 5% CO₂. Diphenyleneiodonium chloride (DPI) and Ribociclib were purchased from Sigma-Aldrich, Inc.

Fiorillo M., Sotgia F, & Lisanti M.P. (2019). “Energetic” Cancer Stem Cells (e-CSCs): A New Hyper-Metabolic and Proliferative Tumor Cell Phenotype, Driven by Mitochondrial Energy. Frontiers in Oncology, 8, 677.

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Neutrophil NET Formation Assay

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Neutrophils were seeded in a 96-well plate and incubated with F. magna strains ALB8, 312 or 505 at an MOI of 20 or with F. magna proteins FAF and L, 3.8 and 3 μg/ml respectively. RPMI 1640 medium was used as a negative control. After 2 h, extracellular DNA was stained with the cell-impermeable dye Sytox Green (Thermo Fisher Scientific, Rockford, IL, United States), which is commonly used to quantify NETs (Carmona-Rivera and Kaplan, 2016 ). The dye was added to the supernatant of the samples for 5 min and fluorescence was detected at wavelength recommended by the manufacturer 504/523 nm. The following inhibitors were used: actin polymerization inhibitor Cytochalasin D (20 μM; Thermo Fisher Scientific, Rockford, IL, United States), store-operated Ca²⁺ entry inhibitor 2-Aminoethoxydiphenyl borate (2-ABP, 50 μM; Sigma-Aldrich, St. Louis, MO, United States), MAPK/ERK kinase U0126 (50 μM; Sigma-Aldrich, St. Louis, MO, United States) and inhibitor of nitric oxide synthetase diphenyleneiodonium chloride (DPI, 10 μM; Sigma-Aldrich, St. Louis, MO, United States).

Neumann A., Björck L, & Frick I.M. (2020). Finegoldia magna, an Anaerobic Gram-Positive Bacterium of the Normal Human Microbiota, Induces Inflammation by Activating Neutrophils. Frontiers in Microbiology, 11, 65.

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Immunofluorescence Staining of Rabbit Neutrophils

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Rabbit anti-human Rab27a polyclonal antibody (polyAb), mouse anti-α tubulin monoclonal antibody (mAb) was purchased from Sigma (St. Louis, MO). Mouse anti-human CD11b (complement receptor 3, CR3) mAb for flow cytometry was purchased from DAKO (Glostrup, Denmark). Rabbit anti-human MPO polyAb, rabbit anti-human histone H3 mAb (D1H2) and mouse anti-human histone H4 mAb (L64C1) was from Cell Signaling Technology (Danvers, MA). Rabbit anti-human histone H4 (citrulline 3) polyAb was from Millipore (Billerica, MA). Texas Red-conjugated zymosanA, 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H₂DCFDA) and Sytox Green were purchased from Lifetechnologies (Carlsbad, CA). Puromycin, phorbol 12-myristate 13-acetate(PMA) and diphenylene iodonium chloride (DPI), an NADPH oxidase inhibitor were purchased from Sigma (St. Louis, MO) and RPMI1640 medium was from Wako (Osaka, Japan). Penicillin-streptomycin mixed solution was from Nacalai Tesque (Kyoto, Japan), and polybrene was purchased from Millipore (Bedford, MA). Aminophenyl fluorescein (APF) and hydroxyphenyl fluorescein (HPF) were purchased from Sekisui Medical (Tokyo, Japan). Hoechst33342 was purchased from Dojindo Laboratories (Kumamoto, Japan). DNaseI was purchased from Wako (Osaka, Japan).

Kawakami T., He J., Morita H., Yokoyama K., Kaji H., Tanaka C., Suemori S.I., Tohyama K, & Tohyama Y. (2014). Rab27a Is Essential for the Formation of Neutrophil Extracellular Traps (NETs) in Neutrophil-Like Differentiated HL60 Cells. PLoS ONE, 9(1), e84704.

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Detecting Protein Oxidation in Cells

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OxyBlot™ protein oxidation detection kit was purchased from Merck Millipore (EMD Millipore, Billerica, MA, USA). WST reagent was from Dojindo (Kumamoto, Japan). SB203580 was obtained from Calbiochem (San Diego, CA, USA). Rhodamine-conjugated donkey anti-mouse IgG was from Santa Cruz Biotechnology (Dallas, TX, USA). Antibodies against β-tubulin, GAPDH, phospho-p38, p-ERK, TXNIP, as well as horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgG were obtained from Cell Signaling Technology (Danvers, MA, USA). NOX2 was bought from Bioss (Tokyo, Japan). DMNQ was from Nacalao Tesque (Kyoto, Japan). EZ blue gel staining reagent, LPS, Lindane, apocynin, diphenyleneiodoniumchloride (DPI), Menadione, anti-Cx43, anti-β-actin, and all other chemicals were from Sigma (Tokyo, Japan).

Huang Y., Mao Z., Zhang X., Yang X., Sawada N., Takeda M, & Yao J. (2019). Connexin43 Is Required for the Effective Activation of Spleen Cells and Immunoglobulin Production. International Journal of Molecular Sciences, 20(22), 5789.

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