Novel flow cytometric analysis of the blood–brain barrier

Abstract

The blood–brain barrier (BBB) is primarily comprised of brain microvascular endothelial cells (BMVEC) and astrocytes and serves as a physical and chemical barrier that separates the periphery from the brain. We describe a flow cytometric method using our in vitro model of the human BBB to characterize BMVEC surface junctional proteins critical for maintenance of barrier function, cell viability, and leukocyte adhesion. For this methodology, BMVEC are cocultured with astrocytes in a transwell tissue culture insert to establish the barrier, after which time the BBB are treated with specific agents, and the BMVEC collected for flow cytometric analyses. We use a standard and optimized method to recover the BMVEC from the coculture model that maintains junctional protein expression and cell viability. A novel leukocyte adhesion assay enables a quantitative analysis of peripheral blood mononuclear cell (PBMC) interactions with the BMVEC and can be used to assess the adhesion of many cell types to the BBB. Furthermore, this method enables the concomitant analysis of a large number of adhesion molecules and tight junction proteins on both the BMVEC and adherent PBMC under homeostatic and pathologic conditions. Flow cytometry is an extremely powerful tool, and this technique can also be applied to assess variables not performed in this study, including cell cycle progression, and calcium flux.

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“…Our in vitro transwell model of the human BBB model was made as previously described [ 85 – 91 ]. Briefly, astrocytes (1 × 10 5 cells/insert) were seeded on the underside of a tissue culture insert comprised of a polycarbonate membrane with 3 μM pores (Falcon, Corning, NY) and allowed to adhere for four hours at 37 °C, 5% CO 2 while continually being fed in 5–30 min intervals with M199C.…”

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“…MDM and Huh7 cells were recovered from tissue culture plates using TrypLE Express (Invitrogen Grand Island, NY) for 10–15 min at 37°C, 5% CO 2 as previously described (13). After recovery, the cells were washed once with PBS (Gibco) and separated for extracellular and intracellular staining.…”

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“…Labeled proteins (120 μL) were mixed with 1 mL of 10 7 HBMEC in PBS containing 2% FBS for 1 h at room temperature at dark. After mixing, the HBMEC–protein complexes were washed with PBS containing 2% FBS twice. , Finally, the washed complex solution was filtered for FACS. Each experiment was performed in triplicates.…”

“…After mixing, the HBMEC−protein complexes were washed with PBS containing 2% FBS twice. 15,16 Finally, the washed complex solution was filtered for FACS. Each experiment was performed in triplicates.…”