Cardiac fibroblast isolation, TGF-β-induced differentiation, and 15-epi-LXA4 treatment

The mice were anesthetized using 2% isoflurane, and the chest cavity was opened to remove the heart. After heart isolation, the atria, and right ventricle were removed, and the LV was minced using surgical blades. Cardiac fibroblasts (CF) from the LV were isolated by enzymatic digestion with 600 U/ml collagenase II and 60 U/ml DNase I. Cells at passage 2 were plated in 6-well plates (5 × 10⁴ cells/well) and allowed to attach at 37 °C overnight, then washed using DMEM/F-12 media with 10% FBS and 1% antibiotics to remove unattached cells. The cells were plated on Millicell EZ slide-8-well (Millipore). The cells were serum deprived for 24 hrs and then stimulated with TGF-β (15ng/ml) for 18hrs for myofibroblast differentiation and co-incubated with 15-epi-LXA₄ (100 nM). Cells were fixed using 4% PFA (paraformaldehyde), permeated using 0.1% Triton and blocked for 1hr in 10% goat serum. Cells were subsequently incubated with mouse monoclonal anti-α-SMA antibody (Sigma) overnight and Alexa 555-labeled anti-mouse antibody (Molecular Probes), each for 60 min at room temperature. The nucleus was stained using DAPI (Molecular Probes). Cells were mounted using an anti-fade mounting media (Invitrogen) and then visualized and photographed using a Nikon A1 High Speed Laser Confocal microscope. Total 20 cells were counted per field by the morphology i.e., spindle shape vs stellate. Total 5 fields were counted for each group.