Influenza HA expression in Sf9 insect cells. Bar (with pattern) represents the estimated number of cell surface HA molecules in HAS cells. The HA expression level of HAS cells was determined by the number of molecules of fluorescent conjugated antibody HC3 (anti-HA mAb) bound per cell, by flow cytometry. The mean fluorescence intensities (MFI) of the FITC-conjugated HC3 bound to HAS cells, were linearly related to the mean number of mAb binding sites/cell (details in Materials and methods). ∼19% of cells have ∼1.63 × 10³ copies of HA molecules/μm² (n = 5). (Inset a) Confocal image of FITC-conjugated HC3 stained Sf9 cells expressing HA. (Inset b) Stable HA-expressing HAS cells were surface biotinylated and lysed. HA was immunoprecipitated and biotinylated HA was recovered with protein A agarose beads. Biotinylated protein complex was separated by 10% SDS-PAGE and surface HA expression was detected by FITC-conjugated Avidin. Detection of cell surface HA expression in HAS cells indicated that insect cells are suitable for stable expression of influenza HA.