Ebolavirus and Marburg virus GP expression

Vero E6 cells (4 x 10⁵ cells/well, 6-well plate format) were transiently transfected with 2.5 μg of empty or EBOV (variant Yambuku, isolate Mayinga) GP_1,2 or Marburg virus (MARV, variant Mt. Elgon, isolate Musoke) GP_1,2 pCAGGS expression plasmids [21 (link), 22 (link)] using Roche X-tremeGENE HP DNA transfection reagent (Millipore Sigma). Cells were dissociated with enzyme-free cell-dissociation buffer (Thermo Fisher Scientific) 48 h post-transfection (p.t.) and stained with 2 μg/ml of mAb 6D8, followed by Alexa Fluor 488-conjugated goat anti-mouse immunoglobulin G (IgG) antibody (Thermo Fisher Scientific) on ice. As a control, all cells were stained with the same concentration of mouse IgG, followed by the same secondary antibody. These samples were analyzed with an LSRFortessa flow cytometer (Becton, Dickinson and Company, San Jose, CA, USA), and the data were analyzed with FlowJo software (Tree Star, Ashland, Oregon, USA). All experiments were performed in duplicates.