E selectin

Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), and fluorescent dye calcein-AM were purchased from Invitrogen (Carlsbad, CA, USA). DNeasy PowerSoil kit, RNAlater, RNeasy Plus mini kit, QIAzol reagent, QuantiTect reverse transcription kit, qPCR SYBR green master mix, and QuantiTect primers (GAPDH, monocyte chemotactic protein-1 (MCP1), interleukin 8 (IL8), vascular cell adhesion molecule-1 (VCAM1), intercellular adhesion molecule-1 (ICAM1), and E-Selectin) were from Qiagen (Valencia, CA, USA). Protein assay kits were from Bio-Rad (Hercules, CA, USA), and phosphatase and protease inhibitor cocktails were from Sigma-Aldrich (St. Louis, MO, USA). WEHI 78/24 murine monocytic cells were generously provided by Dr. Judith A. Berliner (University of California, Los Angeles, CA, USA).

Total RNA was extracted from the liver (15 mg) and kidney (17 mg) using Animal Tissue RNA Purification Kit (#25700 Norgen Biotek Corporation, Thorold, ON, Canada) according to manufacturer’s instructions. The RNA concentration and purity were measured using a nanodrop (Titertek-Berthold) machine and 200 ng/µL (liver) or 120 ng/µL (kidney) were reverse transcripted. cDNA was prepared using the SensiFAST™ cDNA Synthesis Kit 50 reactions (#BIO-65053 Meridian Bioscience, USA) according to manufacturer’s instructions. Real time qPCR was performed using 100 ng of cDNA (liver and kidney) through the SensiFAST™ SYBR^® No-ROX Kit (#BIO-98005 Meridian Bioscience, USA) according to manufacturer’s instructions. PCR reaction was carried out on the CFX Connect Real-Time PCR Detection System (Bio-Rad). Relative gene expression was obtained after normalization to housekeeping genes (β-actin and GAPDH) using the formula 2^-ΔΔCT as previously described (36 (link)) and folds change was determined by comparison to Sham group. The QuantiTect primers β-actin (#QT00095242, Mm_Actb_1_SG), GAPDH (#QT01658692, Mm_GAPDH_3_SG), ICAM1 (#QT00155078, Mm_ICAM1_1_SG), VCAM1 (#QT00128793, Mm_VCAM1_1_SG), E-Selectin (#QT00114338, Mm_Sele_1_SG) used in this study were purchased from QIAGEN (Germantown, MD, EUA).