Recombinant human tnfα and ifnγ

Phosphate-buffered saline (PBS), Dulbecco’s modified essential medium (DMEM), fetal bovine serum (FBS), antibiotics (10,000 U/mL penicillin/10,000 μg/mL streptomycin), and 0.25% trypsin-EDTA were purchased from Gibco Life Technologies (Grand Island, NY, USA). PowerUp^TM SYBR Master Mix, Halt^TM protease and phosphatase inhibitor cocktails, and other reagents for quantitative real-time polymerase chain reaction (qRT-PCR) were obtained from Thermo Scientific Pierce (Waltham, MA, USA). Enzyme-linked immunosorbent assay (ELISA) kits and recombinant human TNFα and IFNγ were sourced from R&D Systems (Minneapolis, MN, USA). Primary antibodies, including anti-p-nuclear factor (NF)-κB p65, anti-p-c-Jun (AP-1), anti-p-IκB-α, anti-p-signal transducer and activator of transcription (STAT)-1, and anti-p-p38, were purchased from Cell Signaling Technology, Inc. (Beverly, MA, USA). Other primary antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Equipment, chemicals, and horse radish peroxidase-conjugated secondary antibodies were sourced from Bio-Rad (Hercules, CA, USA). Other chemicals, including 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Human umbilical vascular endothelial cells (HUVECs) were purchased from American Tissue Type Collection. Endothelial Growth Medium (EGM) 2 was obtained from Lonza. AlexaFluor 647-conjugated anti-ICAM1, phycoerythryin-conjugated anti-VCAM1, anti-CXCL10, phycoerythryin/Cy7-conjugated anti-CD14 antibodies and the protein transport inhibitor monensin were purchased from BioLegend. Phycoerythryin-Cy5-conjugated anti-E-selectin and APC-conjugated anti-CD19 antibodies were obtained from BD Biosciences. Qdot 605-conjugated anti-CD3 and Live/Dead fixable violet dead stain kit were from Life Technologies. Recombinant human TNFα and IFNγ, and CXCL10 Quantikine ELISA kit were purchased from R & D Systems.