Mycozap cl

HEK293FT (Thermo Scientific) cells at low passages were grown on petri dishes at 37 °C in a humidified atmosphere with 5% CO2 in Dulbecco’s Modified Eagle’ Medium (DMEM, Thermo Scientific) high glucose with 10% fetal bovine serum (FBS), nonessential amino acids, sodium pyruvate (Thermo Scientific), and antibiotics Mycozap-CL (Lonza). For the expression of targets, cells were plated down 24h prior to transient transfection at a density indicated in each experiment. Transient transfection was carried out using either Lipofectamine^TM 2000 (Thermo Scientific) or FuGENE® HD (Promega). Transfection by both reagents consistently resulted in 75%–80% transfection efficiency, assessed by Flow Cytometry. The cells were used for assay after 48 hours of transfection unless otherwise stated in the figure legends.

HeLa, HaCaT, and HEK293T cell lines (ATCC) were maintained at 37°C in a humidified atmosphere of 5% CO₂ in fully supplemented media containing DMEM with 10% fetal bovine serum (FBS), nonessential amino acids, sodium pyruvate (Thermo Fisher Scientific), and Mycozap-CL (Lonza). Primary cardiac fibroblasts were isolated from neonatal mouse hearts as described (Zhang et al., 2014 (link)). Fibroblasts adhering to the dish during the pre-plating step were enriched for 1 week in DMEM:F12 supplemented with 3.5% FBS and 1x Mycozap-PR (Lonza). Primary glial cells were isolated from neonatal mouse brains as previously described (He et al., 2007 (link); Habas et al., 2013 (link)).