A549 cells, parental or stably expressing CD8 or NCOA7, were seeded in 6-well plates. Unmodified cells were treated with 50 nM bafilomycin A1 for 1 hr prior to infection (inhibition control). Medium was removed and cells were infected in serum-free DMEM with A/Victoria/3/75 at MOI of 30 and incubated for 1 hr at 37°C. Cells were then trypsinized, and both fixed and stained using IntraStain (Dako) with monoclonal antibody A1 (14 (link)), which is specific for HA that has undergone low pH induced conformational change (provided by Yohei Yamauchi). Alexa-647 conjugated anti-mouse total IgG (Life Technologies) was used as secondary antibody, and cells were analyzed by imaging flow cytometry (see below) using the 647 nm laser for excitation.
Protocol detail
Conformational Change Tracking of Viral Hemagglutinin
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A549 cells
Anti igg
Antibody
Antibody a1
Bafilomycin a1
Cells
Imaging cytometry
Infection
Inhibition
Mouse
Parental
Serum
Corresponding Organization :
Other organizations : King's College London, Institut de Recherche en Infectiologie de Montpellier, Université de Montpellier, Centre National de la Recherche Scientifique
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Variable analysis
independent variables
- Parental cells
- Cells stably expressing CD8
- Cells stably expressing NCOA7
- HA conformational change (measured by monoclonal antibody A1 (14 (link)))
- Cells were seeded in 6-well plates
- Cells were infected with A/Victoria/3/75 at MOI of 30
- Cells were incubated for 1 hr at 37°C
- Cells were trypsinized, fixed, and stained using IntraStain (Dako)
- Alexa-647 conjugated anti-mouse total IgG (Life Technologies) was used as secondary antibody
- Cells were analyzed by imaging flow cytometry using the 647 nm laser for excitation
- unmodified cells were treated with 50 nM bafilomycin A1 for 1 hr prior to infection (inhibition control)
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