Apc anti mouse ifnar1

Cells were cultured, left uninfected or DENV-infected, as described above. At 24 h post infection, cells were harvested, blocked in phosphate-buffered saline with 3% (w/v) bovine serum albumin and immunostained with control IgG (APC Mouse IgG1, cat 3400119, BioLegend) or anti-IFNAR1 (allophycocyanin (APC) anti-mouse IFNAR1, cat #127313, BioLegend)-conjugated antibody. Stained cells were fixed and subjected to flow cytometry (Accuri, BD systems, Franklin Lakes, NJ, USA). Staining was performed in triplicate and repeated on n=2 independent pMEF isolations.

Cells were stained for flow cytometry using CD3, CD4, and CD8 antibodies as well as dead cell marker for T-cells, and CD86, CD64, and CD11c for dendritic cells and macrophages. T-regulatory cells were fixed and stained using the T reg detection kit Miltenyi Biotec) according to manufacturer’s specification.
Antibodies used for flow were as follows: α-CD4 PE (Miltenyi Biotec), α-CD8 Vivobright FITC (Miltenyi Biotec), α-CD3 APC (MIltenyi Biotec), α-CD11c FITC (MiItenyi Biotec), α-CD86-PE (Miltenyi Biotec), and α-CD64-APC (Miltenyi Biotec).
Danger-Associated Molecular Patterns were analyzed by staining targeted cells with anti-human Calreticulin (CRT)-A647 (Bioss), anti-human HSP70-A647 (Bioss), anti-human GRP94-A647 (Bioss), and anti-human HMGB1-A647 (Bioss). yH2AX was analyzed by staining the cells using anti-human yH2AX (Biolegend). All antibodies were cross-reactive with mice.
To measure immunomarkers, the cells were stained with APC anti-mouse IFNAR-1 (Biolegend), APC anti-mouse CD252 (Biolegend), APC anti-mouse CD54 (Biolegend), APC anti-mouse CD152 (Biolegend), APC anti-mouse CD274 (Biolegend), APC anti-mouse CD137 (Biolegend), APC anti-mouse CEACAM-1/CD66a (R&D), Alexa Fluor 647 anti-mouse B7-H2 (R&D) Alexa Fluor 647 anti-mouse TRAILR1/TNFRSF10A (Novus), Alexa Fluor 647 anti-mouse CD27 ligand/TNFSF7/CD70 (Novus).
All cells were analyzed on a Guava Easycyte 8HT flow cytometer.