2.5. Cells Transfection with mRNA-GFP

HEK293T cells were grown in six-well tissue culture plates (Costar) with modified DMEM medium (Sigma-Aldrich, St. Louis, MI, USA) supplemented with 10% FBS (HyClone) and 50 mg/mL gentamicin. Medium (250 µL) containing either 2 µg of mRNA-GFP in PGS envelope or 2 µg of mRNA-GFP in liposomes from Lipofectamine 3000 (ThermoFisher, Waltham, MA, USA) was added to the wells of a culture plate with a monolayer of 70%–80% confluent cells. The control well was injected with 2 μg of “naked” mRNA-GFP. The cell plate was placed in a 37 °C CO₂ incubator and incubated for 4 h. After that, the culture medium was replaced with a fresh one and the incubation continued for 24 h. The results were visualized using an Olympus CKX53 microscope or flow cytometry. To determine GFP expression levels, 20,000 events per sample gated on single cells were acquired on a Ze5 flow cytometer (Bio-Rad Laboratories Inc., Hercules, CA, USA) and analyzed using FlowJo software.