Pe cy7 conjugated mouse anti cd11b clone icrf44

Surface immunostainings were performed at day 6 post-isolation of the CD14 + cells from PBMCs. After a 15 min-incubation step with human Fc blocking reagent (MACS Miltenyi Biotec), cell surface markers were detected by a 30 min-incubation at 4 °C with 4 µg/mL Pacific Blue-conjugated mouse anti-CD14 (clone 63D3, Biolegend), a 1:20 dilution of APC-conjugated mouse anti-CD163 (clone REA812, MACS Miltenyi Biotec), 0.14 µg/mL PE-conjugated mouse anti-HLA-DR (clone LN3, Invitrogen) and a 1:20 dilution of PE-Cy7-conjugated mouse anti-CD11b (clone ICRF44, BD Biosciences), diluted in staining buffer (PBS - 1% FBS). Flow cytometric analysis was performed using a BD FACS Canto II and the data were analyzed with Flow Jo software (Tree Star).

The antibodies used for immunostaining were mouse anti-E glycoprotein (4G2) kindly provided by P. Despres (PIMIT, Université de La Réunion-INSERM France) and anti-Mouse IgG (H + L) Alexa Fluor 555 Secondary Antibody (Life Technologies). The antibodies used for western blot detection were anti-ISG15 (Santa Cruz), anti-flag (clone M2; Sigma-Aldrich) and mouse anti-actin (clone AC74; Sigma-Aldrich). The antibodies used for FACS analysis were APC-conjugated anti-CD163 (clone REA812, MACS Miltenyi Biotec), PE-conjugated mouse anti-HLA-DR (clone LN3, Invitrogen); PE-Cy7-conjugated mouse anti-CD11b (clone ICRF44, BD Biosciences), Pacific Blue-conjugated mouse anti-CD14 (clone 63D3, Biolegend). Other reagents include TLR3 agonist poly(I:C) (Invivogen); Hoescht (Life Technologies) and cDNA synthesis and qPCR kits (Life Technologies). The reagents for the preparation of the libraries include: Vivaspin unit 100 MWA (Sartorius); Pico Total RNA kit (Agilent), T4 RNA ligase (New England Biolabs); Superscript III (Life Technology); Ultra DNA Library Prep kit for Illumina (New England Biolabs): AMPure XP beads (Agencourt); and High Sensitivity DNA kit (Agilent).