RNA Extraction and Quantification for Cell Types

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.