Flow cytometry analysis of rIgG binding to HA-expressing K530 cell lines was performed essentially as described 31 (link). Briefly, K530 cell lines were thawed from cryopreserved aliquots and expanded in culture for ≥3 days. Pooled K530 cells were incubated at 4°C for 30 min with 0.4 μg/ml recombinant human IgGs diluted in PBS plus 2% fetal bovine serum. After washing, cells were labeled with 2 μg/ml PE-conjugated goat anti-human IgG (Southern Biotech) for 30 min at 4°C. Cells were then washed and analyzed with a BD FACSymphony A5 flow cytometer. Flow cytometry data were analyzed with FlowJo software (BD).
Flow cytometry analysis of rIgG binding to HA-expressing 293F cells was performed as follows. 293F cells were transfected using PEI with either plasmid encoding full-length HA from A/Aichi/02/1968(H3N2)(X31) or with empty vector. 36 hours post-transfection, cells were incubated at 4°C for one hour with 0.4 μg/ml recombinant human IgGs diluted in PBS plus 2% fetal bovine serum. After washing, cells were labeled with BB515 mouse anti-human IgG (BD Biosciences) for 30 min at 4°C at the manufacturer’s recommended concentration, followed by washing and fixation in 2% paraformaldehyde. Cells were analyzed with a BD LSRFortessa flow cytometer. Flow cytometry data were analyzed with FlowJo software (BD).
Flow cytometry analysis of rIgG binding to HA-expressing 293F cells was performed as follows. 293F cells were transfected using PEI with either plasmid encoding full-length HA from A/Aichi/02/1968(H3N2)(X31) or with empty vector. 36 hours post-transfection, cells were incubated at 4°C for one hour with 0.4 μg/ml recombinant human IgGs diluted in PBS plus 2% fetal bovine serum. After washing, cells were labeled with BB515 mouse anti-human IgG (BD Biosciences) for 30 min at 4°C at the manufacturer’s recommended concentration, followed by washing and fixation in 2% paraformaldehyde. Cells were analyzed with a BD LSRFortessa flow cytometer. Flow cytometry data were analyzed with FlowJo software (BD).
