Endogro complete medium

The hCMEC/D3 cell line kindly given by Cochin Institute (Paris, France). were used from passages 27 to 33 and cultured with the EndoGRO complete medium (Merck), supplemented with 5% FBS,1% streptomycin-penicillin (Gibco, Carlsbad, CA, United States), and 1 ng/mL b-FGF (Sigma) under 5% CO₂ and 37°C as previously described (Luo et al., 2019 (link)). Culture flasks and plates were pre-coated with 150 μg/mL rat tail collagen type I. Assessment of intracellular calcium kinetics was carried out on confluent cultures in 24-well plates seeded at 5 × 10⁴ cells/cm².

Human brain microvascular endothelial cells (hBMECs), provided from Cell Systems (Kirkland, WA, USA), were cultured in EndoGRO Complete Medium (Merck Millipore), plated in 24-well plates on glass coverslip (5 × 10⁴ cells per well) and incubated for 4h in a 500μL of medium with/without MOLNBs and OLNBs (dilution 1:100 and 1:200) internalized with 6-Coumarine (Sigma-Aldrich) in a humidified CO₂/air-incubator at 37 °C. Fixing was carried out by adding 500 μL of cold paraformaldehyde (PFA, 4%) and by incubating for 15 min at room temperature and rinsing the excess PFA with sterile PBS. After fixing, 4′,6-diamidino-2-phenylindole (DAPI) and Rhodamine-Phalloidin (R415, Invitrogen™, Thermo Fisher Scientific, MA, USA) staining reactions were performed to label cells nuclei and the actin filaments. Fixed cells were kept at 4 °C for 24 h and fluorescence images were acquired by a confocal laser scanning microscope (LSM 900, Carl Zeiss, Oberkochen, Germany) equipped with a 40X oil immersion objective, obtaining a field view of at least 5 cells. A wavelength of 505 nm was used to detect MOLNBs and OLNBs, of 565 nm and 460 nm to detect respectively the actin filaments and the nuclei. Images were processed using the software ImageJ (https://imagej.nih.gov/ij/).