Alexa fluor 488

To measure activation of cultured mouse endothelial cells (bEnd.3), cells were seeded into 12‐well plates and exposed to recombinant murine uPA‐PAI‐1, TNF (100 ng/ml), or saline for 6 h at 37°C. Cells were collected and resuspended in saline before they were then immunostained using antibodies (0.25 μg in 100 µl PBS) directed against ICAM‐1/CD54 (Alexa Fluor 488, BioLegend), VCAM‐1/CD106 (APC, BioLegend), primary anti‐uPA (Santa Cruz Biotechnology, Santa Cruz, CA/USA), or primary anti‐PAI‐1 antibodies (Abcam, Cambridge, UK) followed by incubation with secondary Alexa Fluor 488‐linked antibodies (Invitrogen, Carlsbad, CA). In separate experiments, bEnd.3 endothelial cells were co‐cultured for 6 h with RAW macrophages, which were, prior to this, exposed for 3 h to recombinant mouse TNF, uPA‐PAI‐1 (100 ng/ml), or saline. After washing the samples twice in PBS, samples were resuspended in 200 μl PBS and analyzed by multi‐channel flow cytometry.

For immunofluorescence and/or flow cytometry the following antibodies were used: GK1.5 (anti-CD4), MP33 (anti-CD45), MECA-367 (anti-mucosal addressin cell adhesion molecule-1 [MAdCAM-1]), and ERMP-12 (anti-CD31), which were purified from hybridoma cell culture supernatants by affinity chromatography with protein G-Sepharose (Pharmacia Biotech, Uppsala, Sweden) and labeled with Alexa Fluor-488, -555, or -647 (all from Invitrogen Life Technologies, Breda, the Netherlands). 145–211 (anti-CD3e, eBioscience, San Diego, CA, USA) Alexa Fluor-555 or Pe-labeled, and TUJ1 (anti-neuronal class III β-tubulin, BioLegend, Dedham, MA, USA) Alexa Fluor-488 labeled. Phage-display-derived single-chain antibodies vesicular stomatitis virus-tagged GD3A12 [11 (link)] and HS4E4 [12 (link)] were used and detected by a Cy3-conjugated secondary antibody anti-vesicular stomatitis virus, P5D4 (Sigma-Aldrich, St. Louis, MO, USA). To assure specificity of the antibodies, conjugate-alone controls as well as control serum (rat or rabbit) as replacement of the primary incubation were used. For western blots an anti-mouse decorin rabbit polyclonal was used at 1:1,000 dilution (immunogen: rat decorin; kindly provided by Åke Oldberg).

Fluorescence minus one (FMO) tubes, rainbow bead tubes, and single antibody tubes were prepared as gating references. Antibodies were pipetted into flow cytometry tubes according to manufacturer’s instructions. Antibodies used include CD34 = Brilliant Violet 421 (BioLegend, San Diego, CA, United States), CD45 = Alexa Fluor 488 (BioLegend, San Diego, CA, United States), CD133 = PE (Miltenyi Biotec, North Rhine-Westphalia, Germany) CD31 = Brilliant Violet 605 (BioLegend, San Diego, CA, United States), CD105 = PE-Cy7 (BioLegend, San Diego, CA, United States), Ghost Dye Red 780 = Tonbo Biosciences, San Diego, CA.
Flow cytometry was performed by blinded scientists on a ThermoFisher Attune NxT (Waltham, MA, United States). Samples were analyzed by blinded scientists using FlowJo software (FlowJo, Ashland, OR, United States).

Cells were seeded in 6-well plates (5 × 10⁵ per well). On the next day, media were replaced with media containing 1% serum and the cells were treated with drugs for additional 24 h. Thereafter, cells were washed in acidic buffer (glycine-HCl 100 mM, pH 3.0) and detached from the plate using trypsin. Finally, cells were washed twice in saline containing albumin (1% w/v) and incubated for 30 min at 4 °C using antibodies to EGFR (clone AY13), HER2 (clone 24D2), and HER3 (clone 1B4C3), which were conjugated respectively to the following fluorophores: Alexa Fluor 488, allophycocyanin, and phycoerythrin (BioLegend Inc, San Diego, CA, USA). Fluorescence intensity was measured using the BD LSR II cytometer (BD Biosciences, San Jose, CA, USA) and analyzed by BD FACS Diva software (BD Biosciences).

BNN27 (100 nM), NGF (100 ng/ml), or BDNF (100 ng/ml) were added to cultures after 6 days CGNs in vitro and kept for an additional 16 h. During this period, CGNs were switched to KCl-free and serum-free medium. Finally, the culture was fixed with 4% Paraformaldehyde (PFA), permeabilised with 0.3% triton and blocked for non- specific epitopes with 5% Horse Serum (HS). Staining for β- tubulin+ was performed prior to TUNEL staining. The primary antibody (Cat. No. 801201, Biolegend) was incubated overnight at 4°C and the secondary anti-rabbit Alexa Fluor 488 (Cat. No. A21206; Invitrogen) for 1 h at RT. Subsequently, TUNEL was assessed using a kit from Roche following the manufacturer’s instructions and cultures were analyzed using a fluorescent Zeiss microscope. The number of TUNEL-positive cells co- localized with b-tubulin staining (yellowish nucleus) in each culture was counted using an objective (x40) from 10 visual fields for every condition. The percentage of TUNEL+ β- tubulin+/β- tubulin+ was calculated and the mean number was estimated for each condition from three independent experiments.