Luciferase assay protocol

NFAT-luciferase mice _ENREF_9^{9 (link)}, kindly provided by Dr. Jeffery D. Molkentin (Cincinnati Children’s Hospital Medical Center, Cincinnati, OH), carry nine copies of NFAT-binding sites from the interleukin-4 promoter, upstream of the luciferase gene. Primary ventricular cardiomyocyte cultures from neonatal (one-three days old) NFAT-luciferase reporter mice were prepared as described^{54 (link)}. Cell cultures from three separate isolations were used for experiments. Cardiomyocytes were maintained in serum-free medium 24 hours prior to treatment with recombinant Wnt5a, sFRP3 (R&D Systems, Minneapolis, MN, USA) or endothelin-1 (used as positive control, Sigma-Aldrich, St. Louis, MO, USA) for 24 hours, washed twice with PBS and harvested for luciferase activity quantification according to the luciferase assay protocol (Promega, Madison, WI, USA). Luminescence from duplicates was quantified on a Victor 3 1420 Multilabel Counter (PerkinElmer, MA). Cell culture medium was collected for cell viability analyses (ToxiLight, Lonza Group Ltd, Basel, Switzerland) and measurements of sFRP3 release measured by EIAs (R&D Systems; Stillwater, MN, USA).

The acinar cells were infected with adenovirus-NF-κB-luciferase adenovirus (at 10⁷ IFU/ml), and immediately plated on a 24-well plate and cultured with 6 groups of macrophage supernatants. At 24 and 48 h after infection, the cells were collected and washed with ice-cold PBS, lysed using 250 μl Passive Lysis Buffer (Promega, Madison, WI, USA) and centrifuged (13,000 rpm for 10 min at 4°C). Assays for luciferase activity were performed according to the luciferase assay protocol (Promega) and measured using a luminometer (Veritas; Symantec) and GloMax software (Promega).