Hcf av

Cell cultures were performed using primary human cardiac fibroblasts (HCF) (HCF-av, ScienCell Research Laboratories, San Diego, CA, USA) (3 to 5 passages) and PBMC collected from healthy donors and from patients at different times after myocardial infarction (H0, H24, H48, M1). According to the manufacturer’s instructions, HCF were cultured with complete medium (Fibroblast Medium 2 (FM-2) with 5% FBS, 1% Fibroblast Growth Supplement-2 (FGS-2), and 1% penicillin–streptomycin solution, ScienCell Research Laboratories, San Diego, CA, USA) in a 37 °C, 5% CO₂ atmosphere.
The first day, 3 × 10⁵ HCF were seeded in a 6-well plate and grown in FM-2 complete medium. PBMC were thawed and cultured with Gibco™ RPMI (Roswell Park Memorial Institute medium, ThermoFisher Scientific, Waltham, MA, USA) complete medium (RPMI with 10% FBS, and 1% penicillin–streptomycin solution) in a separate 6-well plate during 24 h in the incubator. The medium of HCF was then replaced by DMEM complete medium, and inserts with 0.4 µm pores were placed in each well. 1.5 × 10⁶ PBMC were distributed in the upper chamber of inserts. For each insert containing PBMC (“PBMC well”), a “control well” (insert without PBMC) was performed. After 24 h of coculture, supernatants were collected and stored at − 20 °C, and fresh HCF were stained and analyzed by flow cytometry.