Tnf α quantikine elisa kit

Adipose cells and tissues were lysed in RIPA buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 12 mM deoxycholic acid, 0.5% Nonidet P-40, and protease and phosphatase inhibitors) and proteins were loaded for SDS-PAGE followed by western blotting. Nitrocellulose membranes were incubated with primary antibodies (1:1000 dilution) and successively with the appropriate horseradish peroxidase-conjugated secondary antibodies. Immunoreactive bands were detected by a FluorChem FC3 system (ProteinSimple, San Jose, CA, USA) after incubation of the membranes with ECL Selected Western Blotting Detection Reagent (GE Healthcare, Pittsburgh, PA, USA).
Cytokine antibody array (R&D Systems Inc., Minneapolis, MN, USA) was used to detect the cytokine protein levels in 200 µg of a pool of total homogenate obtained from murine visceral adipose tissue. Cytokine content was determined through densitometric analysis according to the manufacturer’s protocol.
Tnfα levels in sera and cell culture media were measured by murine and human TNF-α Quantikine ELISA kit according to the manufacturer’s protocol (R&D Systems Inc.).

On day 7, the inferior vena cava was punctured using a 1 mL syringe previously impregnated with heparin (heparin Choay 25.000 IU / 5 mL), to withdraw an average of 0.7 mL whole blood. Blood was then centrifuged at 825 g at 4°C, and plasma was frozen at -80°C. Levels of adhesion molecules VCAM-1, E-selectin and TNFα were measured in plasma by sVCAM-1/CD106 Quantikine ELISA Kit, E-Selectin/CD62E Quantikine ELISA Kit (R&D system), and TNFα Quantikine ELISA Kit (R&D system). The plates were read by a BIOTEK ELx800 analyzer (BIOTEK, France).

Plasma levels of HMGB1 in mice were measured using an ELISA kit from Chondrex, Inc. according to the manufacturer’s instructions (4 (link)). Plasma levels of TNF-α were measured using a TNF-α Quantikine ELISA kit from R&D Systems (5 (link)). Absorbance at 450 nm (sample) and 630 nm (reference) was measured with a Synergy 2 Multi-Mode Microplate Reader (BioTek).

PHrodo™ Green E. coli BioParticles™ was purchased from Thermo Fisher Scientific (Waltham, MA, United States). Stattic, Fludarabinum, C29, TAK-242 were bought from MCE (Monmouth Junction, NJ, United States). BAY 11-7085, SP610025, PD184352, LY294002, AZD0530 and Y-27632 were purchased from Selleckchem (Houston, TX, United States). Mouse IL-1β, IL-6, TNF-α Quantikine ELISA Kit, Cytochalasin D were bought from R&D Systems (Minneapolis, MN, United States). Mouse myeloperoxidase/MPO ELISA Kit was purchased from MultiScience (Lianke) Biotech Co., Ltd. (Hangzhou, China). p-STAT3 (Tyr705), p-STAT1 (Tyr701), p-p65(Ser536), p-Src (Tyr416), p-Lyn (Tyr507), p-SAPK/JNK (Thr183/Tyr185), p-p38 (Thr180/Tyr182), p-ERK1/2 (Thr202/Tyr204) were bought from Cell Signaling Technology (Beverly, MA, United States). STAT3, STAT1, p65, Src, Lyn, JNK, p38, ERK1/2 were purchased from wanleibio (Shenyang, China). β-actin was purchased from Bioworld Technology (Bloomington, MN, United States). FITC anti-mouse F4/80 antibody (clone BM8), APC anti-mouse CD64 (FcγRI) antibody (clone X54-5/7.1), PE anti-mouse CD16/32 antibody (clone 93), purified anti-mouse CD16/32 antibody (clone 93) and Ultra-LEAF™ Purified anti-mouse CD64 (FcγRI) antibody (Clone W18349F) were purchased from Biolegend (San Diego, CA, United States). Rhodamine phalloidin and DAPI were purchased from Beyotime (Nanjing, China).

NE and TNF-α concentration in heart tissues, perfusate, and cell supernatants were determined using the NE research enzyme-linked immunosorbent assay (ELISA) kit (ALPCO#17-NORHU-E01-RES) and the TNF-α Quantikine ELISA kit (R&D System#RTA00), respectively. The western blotting assays of proteins were performed using standard protocol^{22 (link)}. Antibodies for western blotting assays are available in the Supplementary Methods. Quantitative RT-PCR assay of relative mRNA expression was analyzed using standard real-time PCR protocol according to TAKARA qPCR kits. In brief, total RNA was reverse transcribed using a PrimeScript^TM RT Reagent Kit (TAKARA#RR047A). Real-time PCR were performed with the SYBR Premix Ex Taq II (TAKARA#RR820A) in a LightCycler480 real-time PCR system. Primer sequences for genes are shown in Table S3.