Peripheral blood mononuclear cells (PBMCs) (Sanguine Biosciences) were plated in a 96-well plate at 2.5–5 × 105 cells per well in RPMI medium (Cellgro) supplemented with penicillin/streptomycin (2X) and 10% fetal bovine serum. N-[1-(2,3-Dioleoyloxy)propyl]-N,N,N-trimethylammonium methyl-sulfate (DOTAP) (Roche Applied Science) was used as a liposomal transfection agent. Formulation was carried out by mixing siRNAs and DOTAP at a working concentration of 10 μg DOTAP/μg of RNA, and incubated at room temperature for 15–40 minutes. Cultured PBMCs were treated with the formulated siRNAs and incubated for 16 – 20 h at 37 °C. Human TNFa in the cell culture supernatants was quantified in triplicate by using LEGEND MAX™ ELISA kit with pre-coated plates (BioLegend, Inc.), while human IFNa was measured in triplicate using VeriKine™ Human IFNa ELISA Kit (PBL InterferonSource). Small molecule agonist R-848 (Imgenex) was used as a positive control.
R 848
Manufactured by Novus Biologicals
R-848 is a synthetic small molecule that acts as an agonist for Toll-like receptor 7 (TLR7) and Toll-like receptor 8 (TLR8). It is used in research applications to study the immune response mediated by these receptors.
Automatically generated - may contain errors
Lab products found in correlation
Rpmi medium, by Corning (1 mentions)
N 1 2 3 dioleoyloxy propyl n n n trimethylammonium methyl sulfate dotap, by Roche (1 mentions)
Legend max elisa kit, by BioLegend (1 mentions)
Cl097, by InvivoGen (1 mentions)
Cpg odn 2006, by InvivoGen (1 mentions)
Cl307, by InvivoGen (1 mentions)
Imiquimod, by InvivoGen (1 mentions)
2 protocols using r 848
1
PBMC Transfection and Cytokine Quantification
2
Evaluating TLR Ligand Activation
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TLR ligands used in this study were Pam3CSK (EMC Biochemicals, Cat#: L2000), PAM2CSK (EMC Biochemicals, Cat#: L2020), Poly(I:C) (InivoGen, Cat#: Tlr-pic), Lipopolysaccharide (LPS; Sigma-Aldritch Cat#: L4391), Imiquimod (InvivoGen Cat#: Tlr-imiq), R-848 (IMGENEX, Cat#: IMG-2208), CpG ODN-2006 (InvivoGen, Cat#: Tlr-hodnb), Gardiquimod (Inivogen, Cat#: tlrl-gdqs), Loxorobine (Invivogen, Cat#: tlrl-lox), CL097 (Invivogen, Cat#: tlrl-c97-5) and CL307 (Invivogen, Cat#: tlrl-c307) at concentrations as detailed in the corresponding figure legends. All TLR ligands (with the exception of LPS) were supplied with endotoxin levels <0.001 EU/µg and were made up in sterile endotoxin free water.
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