Duoset elisa antibodies

Plasma was collected via tail-vein puncture in heparin-coated tubes. Fasting cholesterol levels were measured by colorimetric assay (STA-384; Cell Biolabs). Plasma levels of suPAR were measured using R&D DuoSet ELISA antibodies and Ancillary Reagent Kit 2 for development of a sandwich ELISA (DY531, R&D Systems). The ELISA has a detection range of 78 to 5000 pg/mL.

Purified CD11c⁺ BMDC (5 × 10⁴/100 µL) were pretreated with T. suis ESP (50 µg/mL) for 2 h or medium alone. Splenic CD4⁺ cells from OT-II mice were negatively selected using a mouse CD4⁺ T cell isolation kit (StemCell Technologies, Vancouver, BC) and labeled with CFSE, as previously described¹¹⁶ . CFSE-labeled CD4⁺ T cells (2.5 × 10⁵/100 µL) were added to CD11c⁺ BMDC cultures (i.e. T cell-to-BMDC ratio of 5:1) and stimulated with 1 nM OVA_323–339 peptide (AnaSpec, Fremont, CA). After 72 h, cells were harvested and processed for flow cytometry analysis; CFSE dilution was determined in gated CD4⁺ T cells. Supernatants from the co-cultures were collected and assayed for IFNγ, TNFα, and IL-10 by sandwich ELISA using standard protocols and DuoSet ELISA antibodies (R&D Systems, Minneapolis, MN).