Ccl2 antibody

Transwell assay was performed to evaluate VSMC migration in DMEM supplemented with 10% fetal bovine serum (FBS) in the presence or absence of macrophages in a transwell chamber (8-μm pore size; Corning). Briefly, macrophages were seeded into the lower chamber in DMEM medium containing 10% FBS and TAK-242, or CCL2 antibody (100 ng/ml, Santa Cruz, Santa Cruz, CA) that neutralized macrophage-induced migration. VSMCs (10⁴/well) were placed into the upper chamber in DMEM medium or DMEM containing 0.2% bovine serum albumin (BSA).
Recombinant Murine CCL2 (1–100 ng/ml, PeproTech 250–10, Rocky Hill, NJ) was added into the lower chamber. Cells on the filter were removed after 10 hours or 24 hours, and the cells in the bottom chamber were fixed and stained with DAPI. The number of cells that migrated across the filter was counted in five random fields (magnification × 100) using a fluorescence microscope.

THP-1 cells were cultured in RPMI medium 1640 + GlutaMAX from Gibco supplemented with 8% fetal calf serum (FCS), 10 mM HEPES, 1 mM sodium pyruvate (sigma) and streptomycin (50 μg/mL) in a humidified atmosphere of 5% CO₂ at 37 °C. THP-1 cell migration was studied in a modified Boyden chamber assay using a transwell chamber system (Fluoroblock, 8 µM pore size, BD Bioscience, Heidelberg, Germany). 2.5 × 10⁴ cells were seeded in the insert in RPMI medium with 0.1% BSA. Migration towards the stimulus or supernatant derived from HAoSMC conditioned media which was added to the lower chamber, was determined after 14 h. For conditioned media HAoSMC were treated with 100 nM AEA (2 h) and 10 ng/mL IL-1β (3 h). For further analysis supernatant was incubated with 50 ng/mL recombinant human CCL2 (#300-04, Peprotech) or 1:1000 diluted CCL2 antibody (#sc-52877, Santa Cruz).