Sybergreen cells to ct kit

For quantitation of gene expression in tissues, hearts were harvested into RNAlater (Invitrogen), and tissue was homogenized on a Polytron homogenizer (Kinematica). RNA was extracted using the RNeasy Micro kit (QIAGEN), and cDNA was synthesized with SuperScript III reverse transcriptase (Invitrogen) using oligo–deoxy thymidine primers (Promega). Real-time qPCR was performed with Fast Sybergren Master mix (Thermo Fisher Scientific) with the primers described in Table S1. For gene expression analysis of purified populations, cells were sorted on a FACSAria II flow cytometer (BD), and RNA and cDNA were prepared as above (Fig. 7) or with the Sybergreen Cells-to-Ct kit (Thermo Fisher Scientific; Fig. 4). qPCR was performed on a Viia7 PCR system (Thermo Fisher Scientific), gene expression was normalized to Gapdh (Δ cycle threshold [CT]), and values were shown either as target gene mRNA levels relative to Gapdh (2^−ΔCT) or further calculated relative to a comparator (2^−ΔΔCT).

Normal human ventricle CFs (Lonza) were grown in fibroblast basal medium supplemented with fibroblast growth factor B, insulin, gentamycin, and 10% fetal bovine serum. For mouse CFs, hearts were digested in collagenase I/DNase, plated into 6-well tissue culture plates, and grown in DMEM containing 20% fetal bovine serum, 50 U/ml penicillin, and 50 µg/ml streptomycin. CFs were used in experiments between two and five passages of generation. For assays, 10⁴/well CFs were plated into 96-well flat-bottom plates and rested overnight before stimulation with 50 ng/ml LPS (Sigma-Aldrich), 50 ng/ml TNF (PeproTech), or 1 mg/ml CAWS. After 4 h, RNA was extracted, and cDNA was synthesized with the Sybergreen Cells-to-Ct kit (Thermo Fisher Scientific) for qPCR.