FcγR-Dependent MCMV Infection Assay

The BW5147:FcγR-ζ reporter assays were performed as a surrogate test for FcγR activation^{36 (link),37 (link)}. Briefly, the assessment of IgG-dependent activation of the BW5147:FcγR-ζ cells was performed by incubating MCMV-infected cells with serial dilutions of serum in DMEM containing 10% (v/v) FCS for 30 min at 37 °C in an atmosphere of 5% CO₂. To remove non-immune IgG, cells were washed three times with DMEM containing 10% (v/v) FCS before co-cultivation with BW5147:FcγR-ζ reporter cells was performed in RPMI containing 10% (v/v) FCS. The ratio between BW5147:FcγR-ζ and infected cells was 20:1. After 16 h of co-cultivation, supernatants were diluted 1:2 in ELISA sample buffer (PBS with 10% [v/v] FCS and 0.1% [v/v] Tween-20) and mIL-2 was measured by ELISA using the capture antibody JES6-1A12 and the biotinylated detection antibody JES6-5H4 (BD Pharmingen, dilution 1:500).