Quantification of cytokine secretion

Cytokines in supernatants of peritoneal macrophages infected for 5 hours, or BMDM or MEF infected for 24 hours were measured according to the instructions of the manufacturer by ELISA (R&D Systems) or by ProcartaPlex multiplex cytokine assay (Affymetrix). Where indicated, cytokine production was stimulated for 5 hours with Pam3CSK4 (10 μg/ml), FSL-1 (5 μg/ml), ultrapure LPS from E. coli O111:B4 (5 μg/ml), ultrapure flagellin from S. typhimurium (10 μg/ml), imiquimod (5 μg/ml), CpG-rich DNA oligonucleotides ODN 1668 (5 μg/ml) (all purchased from InvivoGen), or poly(I:C) (5 μg/ml) (Calbiochem) instead of infection. Absorbance was measured with a Tecan Infinite M 1000 (Tecan Group), and ProcartaPlex bead fluorescence was measured with a Luminex 100 system (Luminex Corporation).