Studying Lymphocyte Transmigration Across a Microvascular Endothelial Cell Monolayer

Abstract

Source: Schulte-Mecklenbeck, A., et al., Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier. J. Vis. Exp. (2017)

This video demonstrates an experimental investigation of lymphocyte transmigration across a blood-brain barrier (BBB) endothelium model. A monolayer of human brain microvascular endothelial cells (HBMECs) is generated in vitro, and peripheral blood mononuclear cells (PBMCs) containing lymphocytes are added to the monolayer. The cells are then incubated to allow lymphocyte transmigration, and the migrated cells are quantified using flow cytometry.

Protocol

1. Preparation of the Cell Culture Inserts

1. Coating of cell culture inserts
   Important note: Avoid touching the membrane of the cell culture inserts.
   1. Add 100 µL fibronectin solution to each cell culture insert (Figure 1A) and one well of a 96-well flat bottom plate (optical control well). Incubate for at least 3 h at 37 °C. After incubation aspirate fibronectin solution.
   2. Add 100 µL HBMEC suspension to the cell culture inserts and the optical control well. Add 600 µl endothelial cell medium-basal (ECM-b) medium to the lower compartment of the cell culture inserts. Incubate for 3 - 4 days at 37 °C / 5% CO₂ until barrier integrity (Figure 1B) is reached, check cell growth by microscopic evaluation of the HBMEC in the optical control well.
      Note: Cell growth beyond four days is not recommended.

2. Migration Assay

1. Preparation of peripheral blood mononuclear cells (PBMC).
   1. Add 10 mL Roswell Park Memorial Institute (RPMI) medium into a 15 mL centrifuge tube and add 200 µL B27 supplement. Count PBMC and centrifuge cells at 300 x g for 5 min. Re-suspend PBMC to a final concentration of 5 x 10⁶ cells/mL RPMI/B27.
2. Set-up of the migration assay
   1. Aspirate medium from the lower compartment followed by the upper compartment of cell culture inserts containing confluent HBMEC monolayers (Figure 1A). Per donor add 100 µL PBMC suspension each to the cell culture inserts and also to one well of a 24-well plate per (in vitro control).
   2. Add 600 µL RPMI/B27 to the lower compartment of the cell culture inserts and 500 µL to the PBMC of the in vitro control and incubate 6 h at 37 °C / 5% CO_2.
3. Harvesting of migrated PBMC
   1. Take out the cell culture insert using forceps and carefully rinse the bottom with 400 µL phosphate-buffered saline (PBS) without touching the membrane. Discard the cell culture insert.
   2. Add 20 µL flow count fluorospheres (approximately 1,000 beads/µL) to the lower compartment of the cell culture insert as well as to the in vitro control and mix well. Transfer 1 mL of resulting PBMC suspension to flow cytometry tubes.

3. Flow Cytometry

1. Sample preparation
   1. Centrifuge PBMC at 300 x g for 5 min at room temperature.
   2. Prepare antibody solution by adding fluorochrome-conjugated antibodies to 100 µL flow cytometry buffer (PBS/1% bovine serum albumin [BSA]/2 mM ethylenediaminetetraacetic acid [EDTA]) per sample. For the results presented below 1 µL CD4-FITC (cluster of differentiation 4-fluorescein isothiocyanate), 1 µL CD3-PerCP/Cy5.5 (peridinin chlorophyll protein/cyanine 5. 5), 1 µL CD56-PC7 (phycoerythrin cyanin 7), 1 µL CD8-A700 (Alexa fluor 700), and 1 µL CD16-A750 (Alexa fluor 750), were used per sample.
   3. Re-suspend PBMC in 100 µL of the antibody solution and incubate for 30 min at 4 °C.
   4. Add 250 µL flow cytometry buffer and centrifuge at 300 x g for 5 min.

Results

Figure 1: Differential Migration of NK-cell Subsets across Uninflamed and Inflamed HBMEC Monolayers. A. A picture of transwell inserts (left) and illustration of the experimental setup (right). B. Validation of HBMEC barrier functions. Left:...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
PBS	Gibco	14190-094	without CaCl2 or MgCl2
Fibronectin 1mg/mL	Sigma	F1141-5MG	from bovine plasma
HBMEC	ScienCell	1000
HBMEC	Pelobiotech	PB-H-6023
ECM-b	ScienCell	1001-b
FBS	ScienCell	1001-b
Penicillin/Streptomycin	ScienCell	1001-b
Endothelial cell growth supplement	ScienCell	1001-b
Transwell	Corning	3472	clear, 6.5mm diameter, 3.0µm pore size
96-well flat bottom plate	Corning	3596
B27	Gibco	17504-044	50x concentrated
48-well plate	Corning	3526
RPMI 1640	Gibco	61870-010
Flow Count Fluorospheres	Beckman Coulter	7547053
Na-EDTA	Sigma	E5134
BSA	Sigma	A2153
CD3-PerCP/Cy5.5	Biolegend	300430	clone UCHT1
CD56-PC7	Beckman Coulter	A21692	clone N901
CD16-A750	Beckman Coulter	A66330	clone 3G8
CD4-FITC	Biolegend	300506	clone RPA-T4
CD8-A700	Beckman Coulter	A66332	clone B9.11