Orange cmtmr dye

hBMECs were detached from the cell-culture plate and stained with CellTracker™ Green CMFDA dye (C2925, Invitrogen, Waltham, MA, USA). HBVPs and NHAs were detached from the cell-culture plate and stained with blue CMAC dye (C2110, Invitrogen) and orange CMTMR dye (C2927, Invitrogen), respectively. After 24 h, fluorescence images were obtained using the Lionheart FX Automated Microscope (BioTek Instruments, Winooski, VT, USA). These chips were used for the permeability test.

Immortalized human brain microvascular endothelial cells (HBMEC; #1000, ScienCell, Carlsbad, CA, USA) were maintained in endothelial cell medium (#1001, ScienCell). Human brain vascular pericytes (HBVP; #1200, ScienCell) and normal human astrocytes (NHA; CC-2565, Lonza, Basel, Switzerland) were maintained in astrocyte (CC-3186, Lonza) and pericyte medium (#1201, ScienCell), respectively. All cells were cultured at 37 °C in a humidified chamber containing 95% air and 5% CO₂. Before seeding cells into the BBB chip, HBMEC, HBVP, and NHA were stained with CellTracker™ Green CMFDA dye (C2925, Invitrogen), Orange CMTMR dye (C2927, Invitrogen), and Blue CMAC dye (C2110, Invitrogen), respectively. Stained cells were imaged by Lionheart FX Automated Microscope (BioTek Instruments, Winooski, VT, USA).