Ng nitro l arginine methyl ester l name

To quantify NETs, the method of Parker et al. (11 (link)) was adopted. Briefly, neutrophils (5 × 10⁶ cells/well in a 200 μl volume) were suspended in HBSS (Mediatech-CellGro, USA) and seeded in a black 96-well plate (Cayman Chemical, Ann Arbor, MI, USA). The cells were treated with PMA (1 μg ml⁻¹), P. fluorescens (1 × 10⁶ CFU), V. harveyi (1 × 10⁶ CFU), or E. tarda (1 × 10⁶ CFU) for 1, 2, 3, or 4 h. The control cells were untreated. After treatment, the membrane-impermeable DNA-binding dye, Sytox Green (5 μM), was added to the cells, followed by incubation for 5 min. Fluorescence was then quantified as relative fluorescence units (RFU) at 485 nm excitation and 530 nm emission using a fluorescence spectrophotometer (Infinite M1000, Tecan, Switzerland). For NETs inhibition assay, neutrophils were pre-incubated with the following inhibitors for 30 min at 22°C: 100 μM ROS scavenger Trolox (Sigma, St. Louis, MO, USA), 1 mM nitric oxide (NO) inhibitor N(G)-nitro-L-arginine methylester L-NAME (Sigma, St. Louis, MO, USA), or 100 μM MPO inhibitor 4-aminobenzoic acid hydrazide (ABAH) (Calbiochem, San Diego, CA, USA). NETs production and measurements were then performed as above.

Pemafibrate was kindly provided by Kowa Co. Ltd (Nagoya, Japan). Mouse CD31 antibody was purchased from BD Pharmingen (San Jose, CA)(550274). Antibodies of phosphorylated eNOS (Ser-1177)(9571), eNOS (32027) and Tubulin (2144) were purchased from Cell Signaling Technology (Beverly, MA). NG-nitro-l-arginine methyl ester (L-NAME) was purchased from Sigma (St. Louis, MO) (N5751). GW6471 was purchased from Cayman Chemical (11697). Recombinant human FGF21 protein was purchased from R&D system (2539-FG-025). Plasma FGF21 levels were measured by ELISA kit (R & D system)(MF2100) [15 (link)]. Plasma adiponectin levels were determined by ELISA kit (Otsuka Pharmaceutical Co. Ltd.)(410713). Adenoviral vectors expressing mouse full-length FGF21 (Ad-FGF21) were constructed under the control of the CMV promoter [16 (link), 17 (link)]. Adenoviral vectors expressing β-galactosidase (Ad-βgal) were used as controls [18 (link)]. Lipid profiles and plasma glucose were analyzed by enzymatic kits (Wako Pure Chemical Industries, Ltd) (total cholesterol, 439–17501) (triglyceride, 290–63701) (glucose, 439–90901).

For nitrite detection, adherent PEM were stimulated overnight with IFN-γ (100 ng/ml) and stimulated with LPS (100 ng/ml) in the presence of N-Acetyl Cysteine (NAC) (Sigma-Aldrich, St. Louis, MO) or N^G-nitro-L-arginine methyl ester (L-NAME) (Sigma-Aldrich) for 48 hr. 50 μl of supernatant was collected for analysis of NO₂- production using Griess reagents. 50 μl of 1% sulfanilamide in 3% H₃PO₄ was added to 50 μl of supernatant followed by 50 μl of 0.1% napthylethylene dihydrochloride in 3% H₃PO₄ and the wells were read on a spectrophotometer at 550 nM. Sodium nitrite was used a standard at concentrations ranging from 1 μM to 125 μM.
For ROS detection, adherent PEM were stimulated overnight with IFN-γ (100 ng/ml) and then labeled with 10 μM CM-H₂DCFDA (Thermo Fisher Scientific) in PBS for 30 min at 37°C, followed by washing and addition of complete media. Inhibitors were added 30 min prior to addition of LPS, PMA (1 μM), or H₂O₂ (200 μM). 30 minutes later, cells were scraped and immediately analyzed by flow cytometry.

LPS, NG-nitro-L-arginine methyl ester (L-NAME), 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT), hematoxylin, and eosin were purchased from Sigma-Aldrich (St. Louis, MO, USA); PrimeScript™ RT Master Mix and SYBR Green Master Mix were purchased from Takara Biotechnology (Dalian, China). Oligonucleotides were synthesized by Sangon Biotech (Shanghai, China). APAP was purchased from Sangon Biotech (Shanghai, China). Assay kits of alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LDH), and myeloperoxidase (MPO) were purchased from Nanjing Jiancheng Institute of Biotechnology (Nanjing, China). Nitric Oxide Assay Kit and Nuclear and Cytoplasmic Protein Extraction Kit were purchased from Beyotime Institute of Biotechnology (Shanghai, China). TRIzol, Dulbecco's Modified Eagle's Medium (DMED), fetal bovine serum (FBS), penicillin, and streptomycin were purchased from Invitrogen (Carlsbad, CA, USA). All other chemicals and reagents were analytical grade and commercially available.