Gap19

Mouse brain vascular pericyte primary cells (iXCells), mouse endothelial cell line EOMA (ATCC), and mouse macrophage cell line J774 (ATCC) were maintained in RPMI supplemented with 10% fetal bovine serum (FBS) or pericyte media (iXCells) supplemented with 10% FBS and incubated at 37°C with 5% CO₂. For co-culture experiments, endothelial cells and pericytes were co-cultured in the proportion of 2:1 (endothelial cells:pericytes) for 24 hours prior to the experimental treatments.
Cells were seeded (10⁵ cells/well) in 96-well plates 2 hours prior to infection with B. ovis at a multiplicity of infection (MOI) of 100 or 1,000. For co-cultures, endothelial cells and pericytes were seeded at 10⁵ cells/well and 5 × 10⁴ cells/well, respectively, in a 24-well plate, and inoculated with B. ovis (MOI 100) and L. monocytogenes (MOI 5) or stimulated with 10 pg/mL of purified E. coli LPS (eBioscience). Soon after inoculation plates were centrifuged (170 × g) at room temperature, followed by incubation at 37°C with 5% CO₂ for 30 min. The cells were then washed twice with PBS and then further incubated with RPMI supplemented with 0.1 mg/mL of gentamycin. Sterile RPMI was used as negative control. Endothelial cells and co-cultures were treated with the gap junction blocker Gap19 (Tocris), 100 µM in each well, or sterile RPMI as negative control.

Confluent DITNC1 cell monolayers, grown on 12 mm coverslips in 24-well plates, were prepared for wound-healing assays, and then stimulated with Protein A-conjugated Thy-1-Fc or Trail-R2-Fc for 7 h. For experiments with inhibitors, cells were pretreated for 30 min with 100 μM Gap19 (Tocris Bioscience, Bristol, UK). Astrocytes were then fixed and stained as previously described [5 (link)]. Coverslips were mounted on slides with 10% Mowiol-2.5% 1,4-Diazabicyclo [2.2.2] octane and samples were observed by confocal microscopy. Cells were considered polarized when the Golgi apparatus was located in the perinuclear area and oriented towards the cell-free zone within a 120° angle (see scheme in Figure 1C). One hundred cells were monitored per condition, and cell polarization was evaluated as the percentage of cells along the wound border exhibiting polarized Golgi structures.