IL-6 ELISA assay

DC and myoblasts were harvested and transferred (respectively 50x10³ and 10x10³ cells/well) to 96-well plates and incubated with LipoPolySaccharide LPS (1 μg/mL), flagellin (10 ng/mL), zymosan (1 μg/mL), CpG-A (10 μg/mL), imiquimod (0.5 μg/mL), poly (I:C) (1 μg/mL), or Pam3CSK4 (100 ng/mL), all from Invivogen, for 16 hr. IL-6 concentration was measured in culture supernatants using a Human IL-6 ELISA MAX Deluxe, according to the manufacturer’s instructions and performing 3 to 4 technical replicates per condition. The results are expressed in picograms per milliliter (pg/mL). Unless otherwise specified in figures or legends, cytokine concentrations in vitro are presented after background subtraction, obtained with cells incubated with vehicle (DMSO or water). In some experiments the TLR9 antagonist ODN TTAGGG (A151) was added 1 hr before addition of stimuli at the concentration of 1 μmol/L.