Fast start sybr green 1 kit

Total RNA from cells was isolated using the RNeasy mini kit (Qiagen, Hilden, Germany). RNA was reverse transcribed into cDNA using the Omniscript RT kit (Qiagen). RT‐qPCR was performed using LightCycler technology (Roche, Germany) and the Fast Start SYBR Green I kit. The expression of the target molecule was normalized to GAPDH. The primer sets used (all from Sigma‐Aldrich) are listed in Table S1.

RNA from pbMEC, MAC-T, pbMFC and RAW 264.7 was extracted with TRIZOL-reagent (Invitrogen). RNA from boMdM was extracted using the RNeasy Plus Micro Kit (Qiagen) according to instructions as provided in the manual. cDNA preparation (Superscript II, Invitrogen) and real time quantification of the mRNA concentrations with the Fast-Start Sybr Green I kit and the LightCycler II instrument (Roche) were done as detailed in [18 (link)], except that per assay 75 ng of total RNA was used as input. Relative copy numbers were titrated against external standards prepared from dilution series (10⁶–10 copies) of the cloned amplicons. They were also normalized across the different cell types against the amount the input of total RNA used for cDNA generation. Values from the MEC models pbMEC and MAC-T have in addition been separately normalized against copies of the not regulated CLIC1-encoding gene [63 (link)], with similar results as based on RNA input normalization. The RNA yield of from boMdMs was very limited. Hence, these data were normalized against copies from the GAPDH housekeeping reference gene. Sequences of oligo nucleotide primers are listed in Additional file 2.

RNA was reverse transcribed into cDNA using an Omniscript RT kit (Qiagen). RNA concentrations were determined using a Nanodrop spectrophotometer. qPCR was performed using a Fast Start SYBR Green I kit (Roche). The results were quantified by the 2−ΔΔC(t) method, using GAPDH expression levels for normalization. The primer sequences for human primers were as follows: CXCL9 FW: ATCAGCACCAACCAAGGGACT, RV: GCTTTTTCTTTTGGCTGACCTG; CXCL10 FW: ATTTGCTGCCTTATCTTTCTG, RV: TCTCACCCTTCTTTTTCATTGTAG; CXCL11 FW: GAAGGATGAAAGGTGGGTGA, RV: AAGCACTTTGTAAACTCCGATG; TNFSF13B: FW: GGAGAAGGCAACTCCAGTCAGAAC, RV:CAATTCATCCCCAAAGACATGGAC; and GAPDH FW: TGATGACATCAAGAAGGTGGTGAAG, RV TCCTTGGAGGCCATGTGGGCCAT.
The following mouse primers were used: GAPDH FW TGGCATTGTGGA AGGGCTCATGAC, RV: ATGCCAGTGAGCTTGCCGTTCAGC; and IRF1 FW: CCCACAGAAGAGCATAGCAC, RV: AGCAGTTCTTTGGGAATAGG.