Nanodrop spectrofluorimeter

Total mRNA was extracted using the RNAeasy Minikit (Qiagen, Crawley, UK) according to the manufacturer’s protocol. Briefly, EC were collected from the transwell co-culture filters using accutase, lysed and then added to a column. After three washes, mRNA was eluted from the column with water. mRNA concentration was measured using NanoDrop spectrofluorimeter (LabTech) and mRNA was stored at −80 °C. To convert mRNA to cDNA, random primers (Promega, USA) were annealed to 1 μg of mRNA for 5 minutes at 70 °C, after which the following mastermix was added to give a final volume of 30 μl: 10 U Superscript II Reverse Transcriptase (RT), 10 U RNAout RNase inhibitor, 1× Superscript Buffer (all from Invitrogen) and 10 mM dNTPs (Promega). The reaction was run at 37 °C for 1 h, followed by 5 minutes at 95 °C. To analyse mRNA, FAM-labeled E-selectin and SOCS3 primers and VIC-labelled 18S primers, were bought as Assay on Demand kits from Applied Biosystems (Warrington, UK). Samples were amplified in duplicates using the 7500HT real-time PCR machine (Applied Biosystems) and analysed using the software package SDS 2.2 (Applied Biosystems). Data were expressed as relative expression units relative to 18S or as fold change (2–ΔΔCt method).

Total mRNA was extracted using the RNAeasy Minikit (Qiagen, Crawley, UK) according to the manufacturer’s protocol. Briefly, PBMC were first lysed, then added to a column, after three washes, mRNA was eluted from the column with water. mRNA concentration was measured using Nanodrop spectrofluorimeter (LabTech) and mRNA was stored at −80°C. To convert mRNA to cDNA, random primers (Promega, Maddison, USA) were annealed to 1 μg of mRNA for 5 minutes at 70°C, after which the following mastermix was added to give a final volume of 30 μl: 10 U Superscript II Reverse Transcriptase (RT), 10 U RNAout RNase inhibitor, 1X Superscript Buffer (all from Invitrogen) and 10 mM dNTPs (Promega). The reaction was run at 37°C for 1 hour, followed by 5 minutes at 95°C. To analyze mRNA, FAM-labelled SPHK1, SPHK2 and SPNS2 primers and VIC-labelled 18S primers were bought as Assay on Demand kits from Applied Biosystems (Warrington, U.K.). Samples were amplified in duplicates using the 7500HT Real-Time PCR machine (Applied Biosystems) and analyzed using the software package SDS 2.2 (Applied Biosystems). Data were expressed as relative expression units relative to 18S or as fold change (2^^{−deltadelta Ct} method).