T Cell Transmigration Assay with CXCL12

T cells were labeled with CellTracker Orange CMRA (Invitrogen, C34551) for transmigration experiments as previously reported (De Haan et al., 2021 (link)). Briefly, T cells were incubated with 2.5 μM CellTracker Orange CMRA for 30 min. Labeling was terminated by adding AIM-V medium. The T cell suspension was centrifuged and resuspended in fresh AIM-V medium at a final density of 400,000 cells/mL. For transmigration assays, 20,000 T cells were perfused through the lumen of the endothelial vessel (top lane) followed by addition of 800 ng/mL CXCL12 (Peprotech, 300-28A) to the opposite (bottom) lane. The OrganoPlate was placed on the OrganoFlow rocker (7° inclination, 8 min interval) in the incubator (37°C, 5% CO₂) and migration was tracked at 0, 24 and 48 h after T cell addition using the ImageXpress Micro Confocal High-Content Imaging System (Molecular Devices). For blocking experiments, T cells were pre-incubated with 50 μg/mL anti-VLA4 antibody Natalizumab (MedChem Express, HY-108831) for 45–60 min before addition to the microfluidic chips. The same concentration of Natalizumab was added to the lumen of the HBMEC vessels.