Avidin hrp

Cytokine production was measured after 2 days in cell culture supernatants and serum. ELISA specific for murine IFN-γ (R&D Systems, Minneapolis, MN, USA) was performed, according to the manufacturer’s instructions. Biotinylated detection Abs were used, followed by avidin-HRP (Biolegend, San Diego, CA, USA). All absorbance reads were made at 450 nm, using a 96-well plate spectrophotometer (VERSAmax, Molecular Device, San Jose, CA, USA).

Macrophages were infected as described under “Bacterial Growth within BMDMs” with a couple exceptions. Macrophage monolayers were infected at an MOI of 1, unless otherwise stated, and following the 2 hour infection, extracellular bacteria were removed by washing macrophage monolayers three times with 1XDPBS (Cytiva/HyClone). Following the third wash, fresh BMDM media (without antibiotics) was added and the plates were placed at 32°C with 5% CO₂. Secreted levels of IFN-β were measured from BMDM supernatants twenty-four hours following the start of the infection following the Sandwich ELISA protocol published by BioLegend. Anti-IFN-β capture antibody (BioLegend, SanDiego, CA) was diluted 1:200 in Carbonate Coating Buffer [0.84% w/v NaHCO₃ (Sigma) and 0.356% w/v Na₂CO₃ (Sigma) dissolved in ddH₂0, pH = 9.5], while the detection antibody (Biotin anti-mouse IFN-β; BioLegend) and Avidin-HRP (BioLegend) were diluted 1:200 and 1:500, respectively, in blocking buffer. Absorbance was measured at a wavelength of 405 nm and secreted levels of IFN-β were calculated from blank corrected data based on absorbance of the IFN-β standard at known concentrations.

Concentrations of IL-33, IL-9, and IFN-γ in culture supernatants were detected by ELISAs as previously described (12 (link)). IL-33 capture/detection Abs were purchased from R&D Systems. Recombinant mouse IL-33 (aa109–266) (ELISA standard) was purchased from R&D Systems. Capture/detection Abs for IL-9 and IFN-γ were purchased from BD Biosciences. Recombinant mouse IL-9 and IFN-γ used as the standards in ELISAs were purchased from R&D Systems and BD Biosciences, respectively. Avidin-HRP was purchased from BioLegend.

Briefly, 96-well ELISA plates (Costar, Corning) were coated with goat anti–human Ig (SouthernBiotech) in PBS at 4°C overnight, then blocked with 1% BSA blocking buffer at room temperature for 1 hour. The culture supernatants and serially diluted standards were then added and incubated at room temperature for 1 hour. After washing, biotin-conjugated anti–human IgG (SouthernBiotech) Ab was added and incubated at room temperature for 1 hour. After washing, avidin-HRP (BioLegend) was added to the plates, incubated for 30 minutes, and visualized with TMB substrate reagents (Invitrogen, Thermo Fisher Scientific). OD₄₅₀–OD₅₇₀ was measured by a spectrophotometer (Bio-Rad). The amount of Ig in duplicate wells was calculated based on the standard.

RPE cells (1.2 × 10⁵) were infected with HCMV Merlin GFP or HCMV Merlin dUL11 GFP at a MOI of 1.0. After 6 days in culture, the cells were trypsinized and washed with ice-cold PBS before lysis in 200 μL 5× SDS-PAGE loading buffer (300 mM TRIS-HCl, 10%SDS, 0.1%bromophenol blue, 50% glycerol, 300 mM β-mecaptoethanol). The lysates were separated via SDS-PAGE before transfer onto a nitrocellulose membrane. The membranes were blocked with Roti block (Roth), and cmvIL-10 was detected using Viral HCMV IL-10 Biotinylated Antibody clone BAF117 (R&D Systems) and Avidin-HRP (Biolegend) prior to visualization using the Super Signal West Femto Maximum Sensitivity Kit (Thermo Scientific).

Concentrations of IL-9 in culture supernatant were detected by ELISA as previously described [23 ]. IL-9 Capture/detection antibodies were purchased from BD Biosciences. Recombinant mouse IL-9 used as the standards in ELISA were purchased from R&D Systems. Avidin-HRP was purchased from Biolegend.