Fitc conjugated 40 kda dextran

Cells were grown on 3 μm pore Transwell filters (Corning) until confluent. After the cells were washed twice with PBS, 100 μg/ml fluorescein isothiocyanate (FITC)-conjugated 40 kDa dextran (Sigma) was added to the apical compartment for 30 min. The fluorescence of basolateral medium was measured using a spectrofluorometer (Thermo Fisher) at an excitation wavelength of 492 nm and detection wavelength of 520 nm.

All procedures involving animals were carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Institutional Animal Care and Use Committee of Temple University (Permit Number: 4179). All efforts were made to minimize suffering. Sprague Dawley (SD) rats were obtained from Taconic Farms (Hudson, NY). In vivo BBB permeability of fluorescein isothiocyanate (FITC) conjugated 40 kDa dextran (Sigma-Aldrich) was measured in two week old SD rats using our previously described protocol [11 (link)]. Briefly, animals were anesthetized with an intraperitoneal (i.p.) injection of a solution of ketamine (87 mg/ml) and xylazine (13 mg/ml) at an appropriate dose (0.5 ml/kg body weight) and a cranial window was created as described previously [11 (link)]. The fluorescent dextran (50 mg/ ml, 1 ml /kg body weight) was injected into the jugular vein through a cannula. The intensity of the fluorescent dextran was measured (Rollera Bolt fluorescence camera from QImaging, Surrey, BC, Canada) in the vessels and brain tissue for 5 seconds every minute and was used to estimate permeability of 40 kDa dextran. At the end of the experiment, the animals were euthanized by overdose of pentobarbitol and then exsanguination.