Journal: PLoS Pathogens

Article Title: Steric Shielding of Surface Epitopes and Impaired Immune Recognition Induced by the Ebola Virus Glycoprotein

doi: 10.1371/journal.ppat.1001098

Figure Lengend Snippet: (A) 293T cells were transfected with empty pCAGGS (vector) or vector encoding GP. Floating and adherent cells were harvested 24 h after transfection, pooled, and stained for GP using the KZ52 antibody, followed by FITC-labeled secondary antibodies, and co-stained for β1 integrin or MHC1 with PE-Cy5 conjugated monoclonal antibodies and assayed by flow cytometry. (B) Following transfection with vector encoding GP, floating 293T cells were removed from adherent cells, stained for β1 integrin and assayed by flow cytometry (left panel). Similarly treated cells were mounted on coverslips, fixed, permeabilized and stained for GP with mouse monoclonal antibodies, followed by Alexa 594 conjugated antibodies and assayed by immunofluorescence microscopy. A representative cell is shown (right panel).

Article Snippet: For detection of AU1 epitopes, cells were stained with rabbit polyclonal anti-AU1 antibodies (Bethyl labs) and detected with FITC goat anti-rabbit IgG (Rockland).

Techniques: Transfection, Plasmid Preparation, Staining, Labeling, Bioprocessing, Flow Cytometry, Immunofluorescence, Microscopy

Journal: PLoS Pathogens

Article Title: Steric Shielding of Surface Epitopes and Impaired Immune Recognition Induced by the Ebola Virus Glycoprotein

doi: 10.1371/journal.ppat.1001098

Figure Lengend Snippet: OV79 SL9 target cells were mock transduced (no Ad) or transduced with Adenoviral vectors expressing GFP (Ad GFP) or GFP and EBOV GP (Ad GP) at an MOI of 300. 48 h after transduction, cells were assayed for GFP expression (A); No Ad = blue trace; Ad GFP = green trace; Ad GP = orange trace. Cells were also stained for MHC1 (B); isotype antibody = shaded peak; No Ad = blue trace; Ad GFP = green trace; Ad GP = orange trace. In parallel, CD8 T cells expressing a transgenic TCR (868TCRwt) that recognizes the SL9 HLA-A2 complex were incubated alone or with mock- (no Ad) or Ad- transduced target cells in a 2∶1 ratio. After co-culture, T cells were surface stained for CD8, then fixed and permeabilized, and stained for 868TCRwt and MIP-1β with APC-H7, FITC, and PE- conjugated antibodies, respectively, and assayed by flow cytometry. (C) CD8 + and 868TCRwt + events were analyzed for MIP-1β staining. (D) Bar graph depicts percent cells positive for MIP-1β, normalized to the No Ad target cell sample.

Article Snippet: For detection of AU1 epitopes, cells were stained with rabbit polyclonal anti-AU1 antibodies (Bethyl labs) and detected with FITC goat anti-rabbit IgG (Rockland).

Techniques: Transduction, Expressing, Staining, Transgenic Assay, Incubation, Co-Culture Assay, Flow Cytometry