H1N1 Hemagglutinin Protein Expression in HEK293 Cells

HEK293 cells (ATCC CRL-1573) were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Paneco) supplemented with 2 mM L-Glutamine (Gibco), 10% fetal bovine serum (HyClone), 50 U/mL penicillin and 50 μg/mL streptomycin (both from Paneco). Transfection of HEK293 cells with H1N1 HA-encoding mRNA was performed as described previously (21 (link)) with minor modification. Briefly, HEK293 cells were plated on a 12-well plate in a density of 2×10⁵ cells per well and maintained at 37°C in 5% CO₂. The next day the medium was replaced with a fresh DMEM without antibiotics and cells were transfected by HA-mRNA using Lipofectamine 3000 reagent (Invitrogen) and Opti-Mem I Reduced Serum Medium (Gibco) in accordance with the manufacturer’s instructions. 24 h after transfection, cells were analyzed by immunocytochemical staining.