A novel flow cytometry-based quantitative monocyte adhesion assay to estimate endothelial cell activation in vitro

Human umbilical vein endothelial cells (HUVECs) were seeded at 1 × 10⁴ cells per cm² in six-well plates. Treatment with TNF-α (2.5, 5 or 10 ng/ml) or lipopolysaccharide (0.5, 1 or 5 μg/ml) treatment was carried out for 12 h to activate the HUVECs, then 5 × 10⁵ fluorescently labeled THP1 monocytes were added and incubated for 30 min. Unbound monocytes were removed by washing with phosphate-buffered saline. Adherent monocytes and HUVECs were dissociated and resuspended in PBS as single cells and quantified by fluorescence using flow cytometry.

Authors: Vincent V, Thakkar H, Verma A, Sen A, Chandran N & Singh A.

View the full protocol on flow cytometry-based monocyte adhesion assays here