The microarray data12 (link) are accessible in the Gene Expression Omnibus database (accession number GSE54181). Plots were generated using the webtool R2: microarray analysis and visualization platform (http://r2.amc.nl ).
Total RNA was isolated using the NucleoSpin RNA II kit (Machery-Nagel, Leiden, The Netherlands) according to the manufacturer’s instructions. Total RNA (0.5–1.0 μg) was reverse transcribed using the SuperScript III First Strand synthesis system from Invitrogen. TaqMan PCR was performed using the TaqMan Universal PCR Master Mix and pre-designed, pre-optimized primers and probe mix for CCL2, RANTES (CCL5), IL-8 (CXCL8), CXCL9 and GAPDH (Applied Biosystems, Foster City, USA). Threshold cycle numbers (Ct) were determined using the CFX PCR System (Bio-Rad, Veenendaal, The Netherlands), and the relative quantities of complementary DNA per sample were calculated using the ΔΔCt method using GAPDH as the calibrator gene.
Enzyme-linked immunosorbent assays (ELISAs) for CCL2, RANTES, IL-8 and CXCL9 were performed according to the manufacturer’s instruction (PeproTech, London, UK).
Total RNA was isolated using the NucleoSpin RNA II kit (Machery-Nagel, Leiden, The Netherlands) according to the manufacturer’s instructions. Total RNA (0.5–1.0 μg) was reverse transcribed using the SuperScript III First Strand synthesis system from Invitrogen. TaqMan PCR was performed using the TaqMan Universal PCR Master Mix and pre-designed, pre-optimized primers and probe mix for CCL2, RANTES (CCL5), IL-8 (CXCL8), CXCL9 and GAPDH (Applied Biosystems, Foster City, USA). Threshold cycle numbers (Ct) were determined using the CFX PCR System (Bio-Rad, Veenendaal, The Netherlands), and the relative quantities of complementary DNA per sample were calculated using the ΔΔCt method using GAPDH as the calibrator gene.
Enzyme-linked immunosorbent assays (ELISAs) for CCL2, RANTES, IL-8 and CXCL9 were performed according to the manufacturer’s instruction (PeproTech, London, UK).
