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4 protocols using azd5904

1

MPO-ANCA Modulation of CD206 Expression

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To investigate effects of muMPO ANCA on CD206 expression, we used a specific MPO inhibitor AZD5904 (MedChemExpress, Shanghai) for cell intervention. BMDMs were kept in medium containing 10% muMPO ANCA positive mice serum treated with or without 20 μM AZD5904 and then harvested for further study. PBMCs were treated with human MPO-ANCA enriched IgG or control IgG at a final concentration of 1 mg/ml and was then incubated with or without 20 μM AZD5904 and harvested for further study. As CD206 is a glycosylated membrane protein, we extracted membrane protein of BMDMs using Mem-PER Plus membrane protein extraction Kit (Thermo Scientific) according to manufacturer’s protocol. The CD206 protein was then detected by western blot. We also performed immunofluorescence for CD206 staining in BMDMs to further validate the results of western blot.
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Investigating Oxidative Stress Pathways

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MPO inhibitor AZD5904, iNOS inhibitor 1400W (MedChemExpress); mitochondria-targeted antioxidant MitoQ (Merck); NOS inhibitor l-NAME, flavoenzyme inhibitor diphenyleneiodonium chloride (DPI), lipopolysaccharide E. coli O111:B4, phorbol 12-myristate 13-acetate (PMA), luminol (all Sigma-Aldrich). L-012 (Fujifilm Wako Chemicals), Hanks’ Balanced Salt solution and phosphate-buffered saline with/without calcium and magnesium (Gibco). The boronate derivative of fluorescein, FlBA, was synthesized as described previously ([14 ]).
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3

Luminol-based Chemiluminescence Protocols

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Luminol (sodium salt), and lucigenin (N,N′-Dimethyl-9,9′-biacridinium dinitrate) were purchased from Sigma-Aldrich (Sigma-Aldrich, MO, USA). L-012 sodium salt was purchased from Wako Chemicals USA (Wako Chemicals USA, Inc., Richmond, VA, USA). Verdiperstat, AZD5904 and hydroxypropyl-β-cyclodextrin (HP-β-CD) were purchased from MedChemExpress (MedChemExpress LLC, NJ, USA). Anti-CTLA-4 (9D9), anti-αPD-1 (RMP1-14), anti-Ly6G (1A8) and polyclonal rat IgG control were obtained from Bio X cell (Bio X cell, NH, USA). Luminol sodium salt was dissolved in sterile phosphate-buffered saline (PBS) to a final concentration of 50 mg/mL or 100 mg/mL and stored at − 20ºC. L-012 powder was dissolved in sterile double distilled water (ddH20) to a final concentration of 20 mM and stored at − 20ºC. lucigenin was dissolved in sterile PBS to a final concentration of 1 mg/mL and made fresh for each imaging session. Verdiperstat and AZD5904 were dissolved in sterile DMSO to a final concentration of 10 mM and stored at − 20ºC; AZD5904 – 5% DMSO, 15% HP-β-CD w/v, 200µL intraperitoneal injection daily, Verdiperstat – 4% DMSO,15% HP-β-CD w/v, 300µL intraperitoneal injection daily.
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4

Reagents for Oxidative Burst Assay

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Luminol (sodium salt), lucigenin (N, N′-dimethyl-9,9′-biacridinium dinitrate), phobol 12-myristate 13-acetate (PMA), G418, casein (from bovine milk, sodium salt), and 4-aminobenzoic hydrazide (4-ABAH, myeloperoxidase inhibitor-1; Calbiochem, EMD Millipore) were purchased from Sigma-Aldrich (Sigma-Aldrich, MO, USA). L-012 sodium salt was purchased from Wako Chemicals USA (Richmond, VA, USA). Verdiperstat, AZD5904, and hydroxypropyl-β-cyclodextrin (HP-β-CD) were purchased from MedChemExpress (NJ, USA). Anti-CTLA-4 (9D9), αPD-1 (RMP1-14), CD8α (2.43), CD4+ (GK1.5), NK1.1 (PR136), and polyclonal rat IgG control were obtained from Bio X cell (NH, USA). Luminol sodium salt was dissolved in sterile phosphate-buffered saline (PBS) to a final concentration of 50 mg/mL or 100 mg/mL and stored at −20°C. L-012 powder was dissolved in sterile double-distilled water (ddH20) to a final concentration of 20 mM and stored at −20°C. 4-ABAH was dissolved in sterile ddH2O heated to 65°C to a final concentration of 2 mg/mL.44 (link) Verdiperstat and AZD5904 were dissolved in sterile DMSO to a final concentration of 10 mM and stored at −20°C; AZD5904—5% DMSO, 15% HP-β-CD w/v, 200 µL intraperitoneal injection daily. Verdiperstat—4% DMSO,15% HP-β-CD w/v, 300 µL intraperitoneal injection daily. Nine grams of casein was dissolved in 100 mL PBS, pH7.2 containing 0.9 mM CaCl2 and 0.5 mM MgCl2.45 (link)
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