Icam1

cDNA was synthesized using 200–1000 ng of total RNA with the superscript II reverse transcriptase (Invitrogen) and subsequently used for qPCR, which was performed with the StepOnePlus™ Real-Time PCR System (Life Technologies) using SYBR Green (Promega). Fold enrichment was calculated using the Delta–Delta CT method normalized to hypoxanthin-guanin-phosphoribosyltransferase (HPRT) as house-keeping reference. Primer for HPRT, Hmox1, Icam1, Vcam1 and IL-1R1 were purchased from Qiagen. Ackr1 primer (forward: 5′-CTT CAC CTT GGG ACT CAG TGT-3′; reverse: 5′-GAC TGG CAG CCC TAA GAG G-3′) were self-designed using Primer Express 3.0 software and were synthesized by Metabion.

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).

Mouse tissues and cultured cells were extracted with TRIzol reagent (Thermo Fisher Scientific), and total RNA concentration and quality was determined with a ND-1000 spectrophotometer (Nano Drop Technologies, Wilmington, DE, USA). cDNA was synthesized from 700 to 1,000 ng RNA using the QuantiTect Reverse Transcription kit (Qiagen, Manchester, UK) according to the manufacturer’s instructions. Real-time quantitative PCR was performed using either Taqman or Sybr Select gene expression master mix (Life Technologies) in the StepOnePlus™ thermal cycler (Applied Biosystems). Primers were purchased from Qiagen (tnfα, il-6, il-12b, ccl2, ccl5, cxcl1, fcγRI, icam-1, βactin) and Taqman (gpr84, βactin). Cycle threshold values were determined by the StepOne software and target gene expression was normalized to housekeeping gene (βactin). Relative expression results were plotted as mRNA expression divided by actin expression, and normalized to basal samples when convenient (24 (link)).