Milliplex map tgf β1 2 3 magnetic bead kit

THP-1 cells were plated as described above 48 h post-infection (hpi). The medium was filtered through a 0.22 µm pore size filter (Millipore, USA) and frozen at –20°C until measurements were made. NO was analyzed using a model 280i NO analyzer (Zysense). For the measurement of cytokines, the samples were stored in 10 mM EDTA, 9 μM aprotinin, and 10 μM E-64 (all from Sigma-Aldrich) at –80°C. IFN-γ, TNF-α, IL-1β, IL-4, IL-6, IL-10, and IL-12p70 were analyzed using the MILLIPLEX^®MAP Human Cytokine/Chemokin Magnetic Bead Panel (Merck Millipore). TGF-β was analyzed with the MILLIPLEX^®MAP TGF-β1,2,3 Magnetic Bead Kit (Merck Millipore). All analyses used the MAGPIX^® system (Merck Millipore), according to the manufacturer’s instructions.

Whole blood was obtained by cardiac puncture in infected and control mice and was collected into tubes containing protease inhibitors, including 10 mM EDTA (Thermo Fisher Scientific, United States), 9 μM aprotinin (Sigma-Aldrich, United States) and 10 μM E-64 (Sigma-Aldrich, United States). The tubes were then centrifuged at 3400 g for 5 min at 4°C, and each serum sample was separated into two 110-μL aliquots, quick frozen in liquid nitrogen and stored at -80°C. These samples were used to quantify the levels of chemokines and cytokines using the following kits according to the manufacturer’s instructions: MILLIPLEX^®MAP Mouse Cytokine/Chemokine Magnetic Bead Panel (Merck Millipore, Germany) for TNF-α, INF-γ, IL-2, IL-1α, IL-1β, IL-9, IL-17, IL-6, IL-5, IL-4, IL-10, KC (CXCL1), MIG (CXCL9), IP10 (CXCL10), MCP-1 (CCL2), MIP-1α (CCL3), MIP-1β (CCL4), RANTES (CCL5), and Eotaxin (CCL11) quantitation and MILLIPLEX^®MAP TGF-β1, 2, 3 Magnetic Bead Kit (Merck Millipore, Germany) for TGF-β1 quantitation in the MAGPIX^® system (Merck Millipore, Germany). All samples were analyzed in duplicate.