Mouse ccl2 je mcp 1

To evaluate SDF-1α, MCP-1, and IGF-1 levels secreted from GECS-conditioned medium (GECS CM), 1052 GECS cells/mm² were plated on poly-HEMA-coated 6-well plates. After 48 h of culture, SDF-1α, MCP-1, and IGF-1 supernatant levels were determined by Mouse CXCL12/SDF-1α, Mouse CCL2/JE/MCP-1, and Mouse IGF-1 immunoassay kits (R&D Systems, Minneapolis, MN, USA). The ELISA reaction product was quantified by measuring absorbance at 450 nm and 540 nm using an iMark microplate reader, and data were analyzed using Microplate Manager v. 6.0 (both from Bio-Rad, Hercules, CA, USA).

Liver tissue was homogenized in lysis buffer (50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1% Triton X, 1 mM NaF, 0.25% Na deoxycholate and protease inhibitors) and the S9 fraction was separated by centrifugation. Protein concentrations of the homogenate were quantified using the BCA method [24] . Monocyte chemoattractant protein-1 (MCP-1), and vascular cellular adhesion molecule-1 (VCAM-1) protein levels were quantified in liver homogenates using the mouse CCL2/JE/MCP-1 and VCAM-1/CD106 Quantikine ELISA kits (R&D Systems, Minneapolis, MN, USA), respectively, after loading equal amounts of protein into each well. Concentrations were normalized to mg of liver protein. Plasma concentrations of MCP-1 were quantified using the mouse CCL2/JE/MCP-1 Quantikine ELISA kit (R&D Systems) following a four-fold dilution.