Duoset sandwich elisa

For the analysis of TARM1 cell-surface expression, BMM and BMDC were stimulated for 24h with the following TLR agonists (all from Apotech): 100 ng/ml Pam3CSK4 (TLR1/2 agonist), 50 μg/ml Poly(I:C) (TLR3 agonist), 100ng/ml LPS from E. coli R515 (TLR4 agonist), 50 ng/ml Flagellin from S. Typhimurium (TLR5 agonist), 80 ng/ml MALP-2 (TLR2/6 agonist), 1 μg/ml Imiquimod (TLR7 agonist), 2 μg/ml CpG ODN 1585 (TLR9 agonist), and 250 ng/ml Profilin from Toxoplasma gondii (TLR11 agonist). For analysis of cytokine secretion, 1 × 10⁵ of BMM or 1.3 × 10⁵ sorted neutrophils were cultured in 96-well tissue culture plates coated with goat anti-rat capture Ab and rat anti-TARM1 crosslinking Ab or a rat IgG_2a isotype control Ab (all at 10 μg/ml). Neutrophils were stimulated in the presence or absence of 10 ng/ml Ultrapure LPS from E. coli K12 (InvivoGen) for 8 h at 37 °C. BMM were stimulated in the presence or absence of either 0.5 μg/ml Pam3CSK4, 0.5 μg/ml PolyI:C, 1 ng/ml LPS, or 10 μM Imiquimod for 16 h at 37 °C. Tissue culture supernatants were collected and cytokine production determined using the Cytometric Bead Array mouse inflammation kit (Becton Dickinson). DuoSet sandwich ELISA (R&D) was used to determine IL-2 secretion by stimulated mouse T cells.