Quantifying HIV-1 Env Surface Expression

Upon transient cotransfection with proviral plasmids encoding green fluorescent protein (GFP) and 90K-myc-encoding constructs, HEK293T cells were harvested after 48 h. Cells were stained with the primary anti-gp120 antibody (2G12) followed by secondary staining with the Alexa Fluor 647-conjugated goat anti-human IgG (Invitrogen) (9 (link), 29 (link)). After the surface HIV-1 Env staining, cells were fixed with paraformaldehyde (PFA) and analyzed for surface HIV-1 Env levels. Due to the disproportional expression of Env inside the cell (high) as opposed to on the cell surface (low) in provirally transfected HEK293T cells (4 (link), 9 (link)), we restricted the analysis to gate R3, which contains cells driving sufficiently high viral gene expression to yield detectable cell surface Env, and normalized these signals to the respective R2 gate. Flow cytometry analysis was performed using FACSCalibur with BD CellQuest Pro 4.0.2 software (BD Pharmingen) and FlowJo V10 software (FlowJo).