The transfection efficiency of brain capillary endothelial cells (BCECs) at different stages of barrier maturity. a Expression of HcRed1-C1 fluorescent protein as seen in BCECs 48 h after transfection. HcRed1-C1 positive cells were expressed at both stages of barrier maturity. The HcRed1-C1 protein distributed to both the cell nucleus and cytoplasm. Scale bar 10 µm. b The relative gene expression of HcRed1-C1 was investigated with RT-qPCR at the two different states of barrier maturity: T−1 (blue) and T1 (green). Non-transfected cells (TCTRL) (purple) showed no HcRed1-C1 gene expression. The gene expression of claudin-5 was included to evaluate the origin of the transfected cells and to measure the degree of tight junction formation at the various stages of barrier maturity. The relative gene expression among the three culture stages was statistically analysed using 1-way ANOVA with Tukey’s multiple comparisons post hoc test (**p < 0.01). Data are presented as sample means ± SEM (n = 6–8). No significant differences were found in the expression pattern of claudin-5 among the three groups. c The transfection efficacy of BCECs was additionally assessed by flow cytometry. A transfection efficacy of about 4% was found in both the immature highly diving stage (T−1) and in the mature non-dividing stage (T1). Results were analysed using the FlowJo V10 software. The cells were gated using forward and side scatter to eliminate cell debris. Additionally, the cells were corrected for auto fluorescence using unlabelled BCECs (purple) to ensure that less than 1% of the HcRed1-C1 positive cells were false positive. Scale bar 10 µm.