Isolation and Culture of Primary Mouse Brain Microvascular Endothelial Cells

Isolation and culture of primary mouse brain microvascular endothelial cells (pMBMECs) from 7–12‐week‐old female mice were performed as previously described (Coisne et al, 2005 (link)). Briefly, mice were euthanized by cervical dislocation, brains were dissected, and meninges, olfactory bulb, brainstem, and thalami were removed. A minimum of six brains per genotype were pooled, homogenized, and resuspended in a 30% dextran (Sigma) solution to obtain a final concentration of 15% dextran. Samples were centrifuged, the vascular pellet was collected, and then digested by incubation with Collagenase/Dispase (2 mg/ml), DNAse I (10 μg/ml) and Nα‐Tosyl‐L‐Lysin‐chlormethyl keton hydrochlorid (TLCK, 0.147 μg/ml) for 30 min at 37°C in a shaker incubator. Digested vessel fragments were then plated in Matrigel (Corning)‐coated 96‐well Nunclon Delta Surface (Thermo Scientific) plates at a seeding density of 51,000 digested capillaries/cm² onto Matrigel‐coated wells. pMBMECs were maintained in Dubelco's Modified Eagle Medium (DMEM, Gibco) with 10% FCS (Biowest), 50 μg/ml of gentamicin (Sigma), and 1 ng/ml of basic fibroblast growth factor (Sigma) at 37°C and 5% CO₂ in a humidified incubator. Once confluent, cells were treated with either mouse recombinant IL‐1β (R&D systems, 10 ng/ml) or vehicle for 24 h.