iPSC-derived BMECs were seeded onto glass bottom plates at a density of 105 cells/cm2 and cultured for 48 hours in EC medium or EC medium conditioned by 3T3s, primary pericytes, or IMR90C4-derived pericyte-like cells. Medium was subsequently replaced with EC medium + 10 μM Alexa 488–tagged 10-kDa dextran. Following 2 hours of dextran incubation, cells were fixed in 4% PFA for 15 min, followed by three washes in PBS. Cells were blocked in 10% goat serum in PBS for 30 min at room temperature. Cells were incubated with anti–Alexa 488 antibody (1:100 in PBS; Life Technologies 11094) overnight at 4°C on a rocking platform. Following three washes in PBS, cells were incubated with Alexa 647 secondary antibody (1:200 in PBS) for 1 hour at room temperature on a rocking platform. Nuclei were labeled with Hoechst, and cells were rinsed three times in PBS. Cells were visualized on a Nikon A1R-Si+ confocal microscope. The lack of permeabilization allows internalized dextran to be visualized only with Alexa 488, while extracellular (surface) dextran is also labeled with Alexa 647.

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