Collagen coated transwells

MDCK cells were grown in collagen-coated transwells (Corning) for 48 h before treatment with DMSO (0.1%) or Bis-T-23 (30 µM; 0.1% DMSO) for 1 h and cisplatin (50 µM) or media for another 23 h. A Millicell ERS Voltohmmeter was used to measure the transepithelial electrical resistance (before and after 24 h of initiating the treatment) in each sample.

Murine brain endothelial cells (MBEC) were isolated as previously described (Assmann et al., 2017 (link)) and maintained in DMEM-F12 (Gibco, ThermoFisher Scientific, UK) supplemented with 10% fetal bovine serum (Sigma, UK), and 1% antibiotic/antimycotic (Gibco, ThermoFisher Scientific, UK), 200 mM L-glutamine (Gibco, ThermoFisher Scientific, UK), 5000 U/ml heparin (Leo Pharma, Denmark) and 30 μg/ml endothelial cell growth supplement (Sigma, UK). The integrity of the endothelial cell monolayer was tested by measuring the diffusive permeability of fluorescein isothiocyanate (FITC)-albumin. Endothelial cells were grown in 6.5 mm in diameter, collagen-coated transwells (Corning, New York, USA) and allowed to form a monolayer. FITC-albumin (1 mg/ml) was then added to the top chamber and after 10 min a sample was taken from the lower chamber to measure basal extravasation. Thrombin (5 IU/ml; Sigma, UK) was then added alone or in the presence of CNP or cANF^4–23 (both 100 nM), and samples from the lower chamber were taken every 15 min for 1 h. Extravasation was expressed as fold change over the basal extravasation (i.e., in the absence of Thrombin).

HUVECs (PromoCell, Heidelberg, Germany) were seeded (3 × 10⁴ cells per well) in endothelial cell growth medium (PromoCell, Heidelberg, Germany) on the filter of collagen-coated transwells (Corning, Kaiserslautern, Germany) for eight days (37 °C and 5% CO₂) until a confluent monolayer was formed. Subsequently, edWB was added to the endothelial cell culture. PBMCs were isolated from blood of healthy donors by density gradient centrifugation (Biocoll separating solution, 1.077 g/mL, Anprotec, Bruckberg, Germany). Cells (1 × 10⁶/^mL) were seeded in RPMI1640 (Anprotec, Bruckberg, Germany)/10% FCS (Anprotec, Bruckberg, Germany) at 37 °C and 5% CO₂.