Flow Cytometry of AR4A Binding

Huh7.5 cells were transfected with HCV RNA as described above, or 293T cells were transfected with J6 E1/E2 expression plasmids in a similar fashion. A total of 48 hours after transfection, cells were treated with ethylenediaminetetraacetic acid and subjected to 2 phosphate-buffered saline washes between the following steps. Cells were fixed in 4% formaldehyde for 10 minutes and incubated for 1 hour with primary antibodies against E1/E2 (AR4A) [20] and E2 (AP33) [31], both at 1 µg/mL. This was followed by 1-hour incubation with the secondary antibodies Alexa Fluor 488 anti-human (ThermoFisher) and allophycocyanin anti-mouse (APC; Biolegend). Cells were resuspended and analyzed on a Becton Dickinson LSR Fortessa equipped with 405-, 488-, and 640-nm lasers and suitable filter sets. Data were collected using FACSDiva 8 (Becton Dickinson) and were analyzed with FlowJo 10 software (FlowJo). For data analysis, live cell populations were gated using front- and side-scatter dot plots and were subsequently analyzed using AP33 (APC) and AR4A (Alexa Fluor 488) density plots. The population of double-positive cells was used to calculate mean fluorescence intensity (MFI) and normalized AR4A fluorescence histograms.