Measurement of intracellular cAMP concentration

Approximately 4.5 × 10⁵ hCMEC/D3 cells per well were seeded in a 24 multiwell plate and grown for 48 h until confluent. Measurement of cAMP levels was performed using the cAMP‐Screen Chemiluminescent Immunoassay System (Thermo Fisher Scientific) according to the manufacturer's instructions with slight modifications as described below. 100 μl of lysis buffer were added per well to the cells and incubated for 30 min at 37 °C with gentle agitation. 90 μl of lysed cell suspension were added to each well of the supplied ELISA 96 multiwell plate. 30 μl of the diluted cAMP‐AP conjugate and 60 μl of the anti‐cAMP antibody were added per well, followed by an incubation for 1 h at 37 °C with gentle agitation. Afterwards the wells were washed three times with 200 μl wash buffer before addition of 100 μl chemiluminescent substrate and incubation for 30 min at room temperature. Luminometric measurement was performed with a Varioskan Flash plate reader (Thermo Fisher Scientific) with a measurement time of 1 s per well. Defined cAMP concentrations served as standard. Chemiluminescence values of treated cell samples were normalized to those obtained from vehicle‐treated cell samples. The results are given as the mean ± SEM from at least six different cell passages.