Quantifying Influenza A Infection Efficiency

A549 cells were obtained from ATCC (ATCC^® CCL-185^TM). A549 and 293FT cells were passaged and cultured in DMEM + l-glutamine, 10% FBS, 1% PenStrep at 37 degrees Celsius, and 5% CO₂ in humidified conditions. PR8 and PR8ΔNS1 stocks were produced and titered as previously described [34 (link)]. Single-round infection assays on A549 cells was performed by infecting cells with PR8 or PR8ΔNS1. Cells were then fixed using the Foxp3/Transcription Factor Staining Buffer Set (Affymetrix, Santa Clara, CA, USA) according to the manufacturer’s protocol. Cells were stained for nucleoprotein using anti-influenza A virus nucleoprotein antibody [431] (FITC) (ab20921) (Abcam), then taken to the flow cytometer for measurement.