Nuclightgreen

A H9C2 cell line stably expressing nuclear restricted eGFP (NucLight Green (Sartorius)) was generated as follows: low passage H9C2 cells were grown to 10% confluence in a 6-well plate and infected with NucLight Green (Sartorius) by adding eGFP-lentivirus particles at an MOI of 1 (100 μL, 10⁶ TU/mL) and Polybrene (8 μg/ml) to 1 mL fresh culture media, followed by incubation for 24 h. The media was replenished and the cells were incubated for a further 48 h. The cells were then detached with trypsin (0.5% in PBS) for 5 min at 37°C/5% CO₂, plated in 6 well plates at 30% confluence in culture media (3mL) and allowed to attach for 24 h. Then, puromycin was added to the media to a final concentration of 3 μg/mL. Drug selection was continued for 5 days. Next, cells were detached with trypsin, resuspended in DMEM media supplemented with 1% FBS (10x10⁶/ml) and the top 10% eGFP-positive cells were collected by fluorescence-activated cell sorting and immediately seeded in a 6-well plate in 3 ml of culture media supplemented with 1 μg/mL puromycin.