Analysis of the cell proliferation of brain capillary endothelial cells (BCECs) from barrier culture days −2 to 3 using a 5-and 6-carboxylfluorescein diacetate succinidyl ester (CFDA SE) assay. BCECs were isolated on day −3 and seeded directly onto inserts. 1 µM CFDA SE was added to the cells at day −2. After 30 min of incubation all the cells were labelled with CFDA SE and the first group were terminated (T−2) (red). Every day for 5 days one group (T−1 to T3) (blue, orange, green, black and purple, respectively) were terminated. The BCECs were microscopically visualised to be confluent on day 0, and subsequently co-cultured with astrocytes and stimulated with hydrocortisone, cAMP and RO-201724. The cells were examined on BD FACS canto™ and analysed with the FlowJo v10 software. The cells were gated using forward and side scatter to eliminate cell debris. Unlabelled BCECs (purple) were used as a negative control.