Mcp 1 instant elisa kit

Plasma free cholesterol, cholesteryl ester, and triglyceride levels were determined in the plasma from all individual mice using enzymatic colorimetric assays (Roche Diagnostics and [21 (link)]). To generate the lipoprotein distribution profiles of the different groups of mice, the remaining plasma from the different mice was pooled per experimental group and subjected to fractionation using a Superose 6 column (injection: 30 µL pooled plasma + 20 µL PBS; 3.2 × 300 mm, Smart-system, Pharmacia, Sweden). Subsequently, the total cholesterol levels were measured in the different lipoprotein fractions using the same enzymatic colorimetric assay as that used for the original plasma samples, taking into account the efficacy of the fast-performance liquid chromatography column. Murine monocyte chemoattractant protein 1 (MCP-1/CCL2) protein levels were assayed in plasma specimens using an MCP-1 instant ELISA kit (eBioscience, Hatfield, UK), according to the manufacturer’s instructions.

Peritoneal exudates were centrifuged at 1500 rpm for 10 min. Cell pellets were resuspended in 1 mL of cell DMEM culture medium containing 10% fetal bovine serum, penicillin/streptomycin, and L-glutamine and subjected to hematological analysis (100 µL volume; Sysmex XT-2000iV Veterinary Heamatology analyzer; Sysmex Corporation, Etten-Leur, The Netherlands) to measure the total leukocyte count. Manual re-gating was performed to identify which fraction of the peritoneal leukocytes was composed of monocytes, macrophages, or SSC^high/FSC^high lipid-laden foam cells, as previously described [24 (link)]. A part of the remaining cell suspension (500 µL) was transferred to 24-well cell culture plates and left to incubate for 24 h at 37 °C in. Subsequently, the medium was collected for the measurement of the protein levels of the pro-inflammatory cytokines MCP-1 and interleukin-6 (IL-6), using, respectively, an MCP-1 instant ELISA kit (eBioscience, Hatfield, UK) or an ELISA MAX™ Deluxe Mouse IL-6 kit (Biolegend, San Diego, CA, USA), according to the manufacturer’s instructions. The residual peritoneal cells (~400 µL cell suspension) were pelleted by centrifugation at 1500 rpm for 10 min and stored at −20 °C in 500 µL of a guanidinium thiocyanate solution for subsequent gene expression analysis.