TLR agonists, CLR agonists, and assay reagents

TLRAs included Pam₃Cys, and polyinosinic:polycytidylic acid (Poly I:C) (TLR1/2, and TLR3, respectively; InvivoGen, San Diego, CA, USA), ultrapure lipopolysaccharide (LPS) from Salmonella minnesota (TLR4; List Biological Laboratories, Campbell, CA, USA), MPLA (TLR4; InvivoGen, San Diego, CA, USA), lipid-based aqueous formulation GLA-AF (TLR4; Infectious Disease Research Institute, Seattle, WA, USA), R848 (TLR7/8; InvivoGen; San Diego, CA, USA), VTX-294 (TLR8; VentiRx Pharmaceuticals, Inc.; Seattle, WA, USA) and CpC ODN 2006 (TLR9; InvivoGen; San Diego, CA, USA). TLRAs were formulated according to manufacturer’s recommendations. CLR agonists (CLRAs) used were Mannan from Saccharomyces cerevisiae (DC-SIGN/MMR; Sigma-Aldrich Co. LLC. St Louis, MO, USA), Hepatitis C Virus E2 (HCV E2; DCIR/BDCA-2; eENZYME LLC, Gaithersburg, MD, USA), biotin glycoprotein 120 (gp120; BDCA-2/DC-SIGN; Immunodiagnostics, Inc., Woburn, MA, USA), alkaline-treated Zymosan (Dectin-1; InvivoGen; San Diego, CA, USA), Curdlan (Dectin-1; InvivoGen, San Diego, CA, USA), Trehalose-6,6-dibehenate (TDB, Mincle; InvivoGen, San Diego, CA, USA), β-glucan peptide (BGP) (Dectin-1; InvivoGen, San Diego, CA, USA), and Whole Glucan Particles (WGP; InvivoGen, San Diego, CA, USA), which were all formulated according to manufacturer’s recommendations. All TLRAs (other than LPS and MPLA) and CLRAs were verified to be free of endotoxin (<1 EU/ml), as measured by Limulus amoebocyte lysate (LAL) assay per the manufacturer’s instructions (Charles River; Wilmington, MA, USA). Sterile Dulbecco’s Phosphate Buffered Saline (DPBS) without Ca2⁺, Mg2⁺, and Phenol Red (Gibco – Life Technologies; Carlsbad, CA, USA) or Roswell Park Memorial Institute (RPMI, Gibco – Life Technologies; Carlsbad, CA, USA) media were included in assays as a negative control when indicated.