Mcp 1 elisa kit

From the mock‐infected or HSV‐1 infected TM cells with or without various treatments, total RNA was extracted using an RNeasy Mini Kit (Qiagen). The total RNA was quantified and reverse‐transcribed to cDNA using PrimeScript RT reagent Kit (Takara Bio). The relative expression levels of mRNAs were determined using a Roche Diagnostics LightCycler 2.0 Real‐Time PCR System (Roche). The sequences of the real‐time PCR primer pairs are shown in Table 1. To ensure equal loading and amplification, all products were normalized relative to β‐actin transcript as an internal control.
Levels of secreted monocyte chemotactic protein (MCP)‐1 were measured using a commercially available MCP‐1 ELISA Kit with pre‐coated plates (R&D Systems). Conditioned medium was harvested, cleared by centrifugation and stored at –70℃. The medium was subsequently acid‐activated and directly assayed using an ELISA plate reader at 450 nm in accordance with the manufacturer's instructions. Protein concentrations were calculated from a standard curve with twofold serial dilutions with the highest standard of 500 pg/ml.

Plasma MCP-1 and insulin levels were determined using commercially available kits (MCP-1 ELISA kit, R&D systems, MN, USA; insulin EIA kit, ALPCO Diagnostics, NH, USA) according to the manufacturers’ procedures. For MCP-1, mouse trunk blood was collected in a heparinized tube. For insulin, the mice were overnight-fasted and then blood was collected from the tail vein. The collected blood was centrifuged at 3,000 rpm for 10 min and the plasma was stored at -80°C until analysis.

Blood glucose levels were determined using a glucometer (ACCU‐Check glucose meter, Roche, MO). Serum cholesterol and FFA levels were measured using Cholesterol Liquicolor (Catalog #1010, STANBIO Laboratory) and Half Micro Test Kit (Catalog #11 383 175 001, Roche), respectively. Serum MDA level was determined using OxiSelect TBARS Assay Kit (Catalog #STA‐330, CELL BIOLABS, CA). Serum Mcp‐1 protein level was measured by a Mcp‐1 ELISA kit (Catalog #DY479‐05, R&D, MN). Serum levels of ALT and AST were measured using ALT/GPT Liqui‐UV kit (Catalog #2930/430, STANBIO Laboratory) and AST/SGOT Liqui‐UV kit (Catalog #2930/2920, STANBIO Laboratory), respectively.

Conditioned media for ELISA were collected by centrifugation at 1500 rpm for 5 min at 4 °C. Human MCP-1 and VEGF concentration was measured, according to the manufacturer’s instructions (VEGF, MCP-1 ELISA kit; R&D Systems, Minneapolis, MN, USA). In brief, all reagents, standard dilutions, and samples were prepared and added to primary antibody-coated well as directed for 2 h at RT, followed by washing four times. Next, the horseradish peroixdase-conjuated secondary antibody solution was treated to each well, and each well was repeatedly washed. Two h later, substrate solution was added. Once the color of the solution changed to blue, the reaction was stopped, and the optical density was measured with the wavelength correction set to 450 nm using an Emax Endpoint ELISA Microplate Reader (Molecular Devices, Sunnyvale, CA, USA). The quantification was completed based on the standard.

ELISA was performed to evaluate MCP-1 protein production in microglial cells, using an MCP-1 ELISA kit based on the sandwich enzyme immunoassay technique (R&D Systems, Minneapolis, MN, USA). The absorbance was determined using a Flex Station 3 microplate reader (Molecular Devices, Sunnyvale, CA, USA).

Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS) and TRIzol reagent were purchased from Invitrogen Life Technologies (Carlsbad, CA, USA). CNP was purchased from Bachem (Torrance, CA, USA). Mouse monoclonal antibody against α-smooth muscle actin (α-SMA) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Mouse monoclonal antibodies against glyceraldehyde 3-phosphate dehydrogenase (GAPDH), extra domain-A (ED-A) fibronectin, collagen I and III, and vimentin were purchased from Abcam (Cambridge, MA, USA). Mouse monoclonal antibodies against von Willebrand factor and troponin I were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Horseradish peroxidase (HRP)-conjugated, rhodamine-conjugated and fluorescein isothiocyanate (FITC)-conjugated secondary antibodies were obtained from BD Biosciences (San Jose, CA, USA). 4, 6-diamidino-2-phenylindole (DAPI) was purchased from Beyotime (Jiangsu, China). Antibodies against ERK1/2 and phospho (p)-ERK1/2 were purchased from Cell Signaling Technology, Inc. (Beverly, MA, USA). U0126 was purchased from Calbiochem (San Diego, CA, USA). An MCP-1 ELISA kit was obtained from R&D Systems (Minneapolis, MN, USA) and the PAI-1 ELISA kit was from American Diagnostica (Greenwich, CT, USA).