Goat anti mouse apc

MRC-5 cells were infected with 1 PFU/cell of wt HCMV and HCMV ΔvFcγR mutants for 72 h and with 10 PFU/cell of HSV-1 wt and ΔgE for 24 h. Cells were resuspended in PBS containing 2 mM EDTA, washed twice in PBS supplemented with 3% (vol/vol) FCS. HCMV infected cells were stained with the F(ab)₂ preparation of Cytotect, goat anti-human-F(ab)₂-Biotin, and Streptavidin-PE (AdB Serotec, UK) or Fcγ fragment-FITC (Rockland Immunochemicals, USA). The comparability of infection of the different HCMV ΔvFcγR mutants was controlled by intracellular staining of CMV nuclear antigens with CCH2 and DDG9 antibodies (Dako, Denmark) and goat anti-mouse-APC (BD Pharmingen, USA) after fixation with 1,5% PFA and permeabilization with PBS supplemented with 3% (vol/vol) FCS and 0,05% (vol/vol) Saponin. HSV infected cells were stained with the F(ab)₂ preparation of Cytotect, goat anti-human-F(ab)₂-Biotin (AdB Serotec, UK) or Fcγ fragment-Biotin (Rockland Immunochemicals, USA) and Streptavidin-APC (Jackson Immunoresearch, USA). After DAPI staining, 1–2×10⁴ living cells were obtained in a FACSCanto II using the FACS Diva software and analyzed with FlowJo (Tree Star Inc, USA).

DCs were harvested at the 5th day after monocyte isolation and were treated with 10 μg/ml TLR2 blocking antibody (#mab2-mtlr2, Invivogen) or isotype control (#mabg1-ctrlm, Invivogen) for 1 h at 37°C at a cell concentration of 2 × 10⁶ cells/ml. Then, the antibody was diluted to 5 μg/ml during DC stimulation at a cell concentration of 1 × 10⁶ cells/ml. As a positive control for TLR2 blocking, cells were stained after 24 h of stimulation with a goat-anti-mouse APC (BD) antibody.

To test the in vivo stability of the reporter viruses MDCK cells were inoculated with lung homogenates at a MOI of 0.1. At approximately 24 hours after inoculation cells were washed twice with PBS-1% FCS. Cells were resuspended in cytofix-cytoperm (BD biosciences) and incubated for 20 minutes on ice. Subsequently they were incubated for 1 hour, on ice, with a monoclonal mouse antibody against NP (IgG2A, Clone Hb65; ATCC), diluted 1/50 in perm-wash buffer (BD Biosciences). Cells were washed once in perm-wash and incubated for 1 hour, on ice, with either polyclonal rabbit-anti-mouse FITC (Dako, Heverlee, Belgium) or goat-anti-mouse APC (BD Biosciences), diluted 1/100 in perm-wash. Cells were washed once with perm-wash, resuspended in PBS and FACS was performed. Data was analysed with FlowJo, version Vx.07 (TreeStar, Inc, Ashland, U.S.A.).