Studying Lymphocyte Transmigration Across a Microvascular Endothelial Cell Monolayer

Take a microplate with transwell inserts coated with ECM proteins to promote cell adhesion.

Add Human brain microvascular endothelial cells, or HBMECs , into the inserts and a growth medium to the lower compartments.

Incubate, allowing the cells to adhere and grow into a monolayer. These cells express tight junctions, mimicking the blood-brain barrier endothelium that limits the passage of cells and molecules.

Remove the medium. Add peripheral blood mononuclear cells, or PBMCs, to the inserts and a culture medium to the lower compartments.

Incubate, allowing PBMC lymphocytes to interact with the endothelial cells and then pass between them, a process called transmigration.

Rinse the insert bottom, collecting the adhered transmigrated cells into the lower compartments.

Introduce fluorescent microbeads as a reference for cell quantification.

Transfer the suspension into a tube, centrifuge, discard the supernatant, and resuspend in a fluorophore-conjugated antibody cocktail to label the lymphocytes.

The transmigrated lymphocytes are ready for quantification via flow cytometry.

To prepare the cell culture inserts for a transmigration assay, first, add 100 microliters of fibronectin sol