The effect of transfection on the integrity of the brain capillary endothelial cells (BCECs). a The experimental design used for in vitro transfection of BCECs. BCECs (yellow) were isolated on day -3, seeded directly onto the inserts. Barrier properties were induced by co-culturing with astrocytes (green) in the presence of hydrocortisone, cAMP and RO-201724. BCECs were transfected at two different stages of barrier maturity: T−1, an immature state, defined by dividing BCECs without barrier properties, and T1, a mature state defined as the BCECs being confluent and having barrier properties. b The integrity of the transfected BCECs (T−1 and T1) (black) was monitored daily by measurements of TEER and compared to non-transfected cells (TCTRL) (grey). Left non-confluent BCECs transfected on day −1 (T−1). No attempts were made to increase tight junction formation. Right on experimental day 1, barrier properties were present (TEER above 150 Ω cm2) in both transfected and control BCECs and lasted for at least 2 days. Significant differences among the two states and their respective controls were analysed using 1-way ANOVA with Tukey’s multiple comparisons post hoc test (***p < 0.001). No significant difference was found between T1 and TCTRL. Data are presented as means ± SEM (n = 34–44). c To investigate the origin of the HcRed1-C1 positive cells, an immunocytochemical analysis was performed for the tight junction protein ZO1. The cells illustrated were transfected at day 1 (T1) and examined 48 h after transfection. The illustrations depict a BCEC containing both the HcRed1-C1 protein and the ZO1 protein (green). Nuclei are counterstained with DAPI (blue). Scale bar 10 µm.