Monocyte chemotactic protein 1 mcp 1

Chemotaxis of T lymphocytes was performed using Boyden chambers. Each chamber consisted of two compartments separated by a membrane. The lower compartment was filled with the stimulant, including conditioned media from M. tb stimulated monocytes and purified IL-8 alone (positive control). Then the membrane (a five-micron pore size polycarbonate filter, Nucleopore, Pleasanton, CA) was placed on the surface and the chamber was assembled. A 200 μL aliquot of the T cell preparation was added to the top of the filter (the top compartment), and the chambers were incubated at 37°C for 4 h. After the incubation, the chambers were centrifuged for 20 min at 1,200 rpm to achieve settling of T lymphocytes, which migrated through the filters, at the bottom of the lower chambers. Then, the filter was removed, and the cells were counted using a hemocytometer (according to the manufacturer's instructions). In some experiments conditioned media from stimulated monocytes were incubated overnight with an antibody against IL-8 (R&D Systems, Minneapolis, MN), or monocyte chemotactic protein-1 (MCP-1) (R&D Systems, Minneapolis, MN), or macrophage inflammatory protein-1α (MIP-1α) (Serotec Inc., Raleigh, NC), or control antibody (mouse IgG1; Sigma, Chemical Co., St. Louis, MO) prior to performing the chemotactic assay.

Glyoxal (GO) and methylglyoxal (mGO) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Lipopolysaccharides from Escherichia coli O111:B4 (LPS) and streptozotocin (STZ) were from Sigma/Aldrich (St. Louis, MO). Monocyte chemotactic protein-1 (MCP-1) was from R&D Systems (Minneapolis, MN). Rho inhibitor CTO4 and Rac activator CN04 were from Cytoskeleton (Denver, CO). Human tumor necrosis factor alpha (TNFα) was from Peprotech (Rocky Hill, NJ). ROS inhibitor, Trolox, and caspase inhibitor, Z-VAD-FMK, were purchased from Selleck Chemicals (Houston, TX).