The overall goal of this transmigration assay is to investigate lymphocyte extravasation into the central nervous system in vitro. This method can help in furthering our understanding of inflammatory diseases of the central nervous system and in the development of new therapeutic approaches. Some advantages of this technique are that it is easily accessible and that it allows analysis of rare lymphocyte populations on single cell level.
This method can provide insight into lymphocyte transmigration across the blood-brain barrier and can be applied to the study of transmigration via the blood-cerebrospinal fluid barrier. In addition, Evans blue permeation analysis can be performed to assess the integrity of the blood-brain barrier model. Begin by covering the bottom of a T-25 cell culture flask with two milliliters of freshly prepared fibronectin solution for an at least three-hour incubation in a cell culture incubator at 37 degrees Celsius and 5%carbon dioxide.
At the end of the incubation, replace the fibronectin solution with 7.2 times 10 to the fourth human brain microvascular endothelial cells, or HBMEC, in six milliliters of complete ECM-b medium, and return the flask to the incubator. When the HBMEC reach an 80%confluency, transfer the supernatant to a 15-milliliter conical tube, and wash the adherent cells three times with five milliliters of PBS. After the third wash, add two milliliters of pre-warmed Accutase solution to the cells for two minutes at 37 degrees Celsius.
Then firmly tap the flask several times, confirming the cell detachment by light microscopy. When the cells begin to lift from the bottom of the flask, use the harvested supernatant to rinse the flask repeatedly until most of the HBMEC are resuspended. Then transfer the cell suspension to a 15-milliliter conical tube for centrifugation, and resuspend the pellet in one milliliter of fresh ECM-b medium for counting.
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.
To test whether barrier integrity has been reached, aspirate the medium from the lower and upper compartments of one cell culture insert, and add 100 microliters of freshly prepared Evans blue solution to the insert. Add 600 microliters of PBS supplemented with B27 to the lower compartment, and return the plate to the incubator. After one hour, use forceps to carefully remove the insert, and transfer 100 microliters of PBS B27 from the lower compartment into two individual wells of a black, polystyrol, 96-well flat bottom plate.
Then load the plate into a multi-mode microplate reader, and determine the optimal z-position. To determine the HBMEC barrier function, measure the excitation of the Evans blue dye solution using the appropriate settings, and compare the acquired data to a standard curve depicting Evans blue permeation across the HBMEC at different time points after seeding. After confirming the formation of the cell monolayer, resuspend the peripheral blood mononuclear cell, or PBMC, sample to a five times 10 to the sixth 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 the 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 one 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 four 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.
When all of the data have been acquired, open the appropriate flow cytometry analysis software. To analyze the NK cell subset transmigration, first select the lymphocytes in a side versus forward scatter channel plot, and display the cells in a CD3 versus CD56 dot plot. Then select the CD56-positive, CD3-negative NK cells.
To distinguish between the transmigrated NK cell subsets, display the cells in a CD56 versus CD16 plot and gate for the CD56bright, CD16dim or negative, and CD56dim, CD16-positive cells. Then select the Flow-Count Fluorospheres from a forward versus side scatter plot. To determine the bead number, display the selected data in a plot of a channel covering an emission wavelength between 525 and 700 nanometers versus time.
Finally, determine the percentage of migrated NK cells as the ratio between the total number of migrated cells and the total number of cells in the in vitro control. Following three to four days of culture, the HBMEC expressed the tight junction molecule Zonula occludens-1 and grow in monolayers that exhibit transendothelial electrical resistance and reduced Evans blue dye permeation. Stimulation of the endothelial cell monolayer with interferon gamma and TNF alpha for 24 hours prior to a whole PBMC transmigration assay results in an increased migration of all of the analyzed lymphocyte populations.
Interestingly, CD56bright, CD16dim or negative NK cells exhibit a higher migratory capacity than CD56dim, CD16-positive NK across both unstimulated and cytokine-activated HBMEC. Although the relative increase of transmigration through an inflamed cell monolayer is higher for the CD56dim cells overall, mimicking the in vivo observations that CD56bright, CD16dim-negative NK cells are enriched in the intrathecal compartment. One mastered, this technique can be completed within 10 hours.
Pretreatment of the in vitro transmigration setup with interferon gamma and TNF alpha enables the study of lymphocyte migration under inflammatory conditions. Our protocol may be modified by applying shear forces or by co-cultivation with other cells of the blood-brain barrier, like astrocytes, to provide a refined model for lymphocyte transmigration into the central nervous system. After watching this video, you should have a good understanding of how to investigate lymphocyte transmigration using an in vitro model of the human blood-brain barrier.
