Anti cd31 alexafluor 647

Confluent cells seeded onto T25 flask were treated for 24 h according to their treatment. Cells were stained in suspension with antibodies against endothelial markers, anti-CD31 AlexaFluor 647 (BD Biosciences) and anti-vWF AlexaFluor 405 (ThermoFisher), according to their respective manufacturer’s instructions. The cells were analyzed by flow cytometry on a FACS-LSRII instrument integrated with FACSDiva software (BD Biosciences). Graphs show median fluorescent values (50,000 events/sample).

For viability assay, cultures were harvested on ice and resuspended in PBS containing 3% FCS and 5 mM EDTA. DAPI staining (Miltenyi) was used at 1/1,000 dilutions 5 min before analysis. For 3D recovery and viability testing, cells were dissociated from recovered spheroids using the BD Recovery Solution (BD Biosciences) following manufacturer instructions.
For endothelial cells characterization an anti-CD31-Alexafluor 647 (BD Biosciences) was used at 1/100 dilution.
For intracellular staining, cells were fixed and permeabilized using a BD Bioscience Fix/Perm assay following manufacturer instructions. Anti-SRC, phospho-SRC and FGF-2 unlabeled primary antibodies (Cell Signaling) were used 30 min at 1/100 dilution and secondary anti rabbit-Alexa Fluor 488 (Cell Signaling) was used at 1/300 for 30 min. A MACSQuant instrument was used to perform experiments and FlowJo 10.4 (Treestar Inc.) software was used to analyze all data.

Mice were anesthetized with sodium pentobarbital by intraperitoneal injection and transcradially perfused with ice-cold PBS. Meninges were quickly removed and digested for 12 min at 37 °C in preheated DMEM (Gibco) with 2% FBS (Gibco), 1 mg/ml collagenase VIII, and 0.5 mg/ml DNase I (Sigma-Aldrich). At the end of digestion, 1 ml DMEM with 10% FBS was added to the solution to terminate digestion. The digested meninges were filtered through a 70-μm nylon-mesh filter. Individual samples consisted of cell suspensions pooled from three meninges and washed with ice-cold fluorescence-activated cell sorting (FACS) buffer (PBS, 1 mM EDTA, 1% BSA, pH 7.2). Cells were then centrifuged at 400g, and 4 °C for 5 min, resuspended in 400 μl ice-cold FACS buffer with anti-CD45-BB515 (BD Bioscience, 564590, 1:200), anti-CD31-Alexa Fluor 647 (BD Bioscience, 563608, 1:200), and anti-podoplanin-PE (eBioscience, 12-5381-82, 1:200), and incubated for 30 min at 4 °C. Cells were washed in ice-cold FACS and sorted by MoFlo Astrios EQ (Beckman Coulter, Indianapolis, IN, USA) and analyzed by the FlowJo software (version 10, Tree Star, Ashland, OR, USA). The LECs were gated as CD45⁻CD31⁺podoplanin⁺ cells.