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 solution to each insert and to one well of a 96-well flat-bottom plate. After three hours at 37 degrees Celsius, replace the fibronectin solution with 100 microliters of HBMEC, and add 600 microliters of fresh ECM-b medium to the lower compartments of the cell culture inserts.

Then, return the plates to the cell culture incubator for three to four days. Resuspend the Peripheral Blood Mononuclear Cell, or PBMC sample to a 5 times 10 to the 6 cells per milliliter concentration in RPMI medium supplemented with B27. Next, aspirate the medium from the lower and upper compartments of the appropriate number of cell culture inserts containing the confluent HBMEC monolayers, and add 100 microliters of PBMC per donor to the cell culture inserts, and to one well of a 24-well plate per in vitro control.

Add 600 microliters of RPMI plus B27 to the lower compartments of the cell culture inserts and 500 microliters to in vitro control wells, and return the plates to the cell culture incubator for six hours. At the end of the incubation, use forceps to remove the cell culture inserts, carefully rinsing the bottoms with 400 microliters of PBS without touching the membranes.

Then, thoroughly mix 20 microliters of flow-count fluorospheres with the cells in the experimental and in vitro control wells, and transfer 1 milliliter of the resulting PBMC suspensions to the appropriate corresponding flow cytometry tubes. Collect the cells by centrifugation, and add the fluorochrome conjugated antibodies of interest in 100 microliters of flow cytometry buffer to each sample. After 30 minutes at 4 degrees Celsius, wash the cells with 250 microliters of fresh flow cytometry buffer and resuspend the pellets in the appropriate volume of flow cytometry buffer for analysis.