Ephb1 fc

Cells were seeded on 8 well chamber slides (Millicell EZ slide, Millipore). At 80% confluence, cells were fixed with 4% paraformaldehyde (EMD Millipore), permeabilized with 0.2% Tx-100 (Pierce), non-specific binding blocked with Superblock blocking buffer (Pierce) and incubated with antibody. Antibody binding was detected with Alexa fluor conjugated secondary antibody (Invitrogen), coverslips mounted with Vectashield plus DaPi mounting medium (Vector Labs) and analyzed with a confocal microscope (Olympus Fluoview 1000).
For surface staining of EphrinB1, cells were again seeded on 8 well chamber slides. When they reached 80% confluence, slides were put on ice for 20 minutes to slow membrane turnover. Cells were incubated with EphB1-Fc (R&D Systems) which consists of the extracellular domain of EphrinB1's cognate receptor (EphB1) fused to human IgG1; this generates a soluble reagent that binds surface expressed EphrinB1. Following washes, cells were then fixed with 4% paraformaldehyde and bound EphB1-Fc detected with anti-human FITC (Sigma, #F9512).

As serum is known to induce astrocyte activation, before treatment primary mouse astrocytes were washed once with HBSS and subsequently serum-starved for a variable time (1–24 h) in Sato’s serum-free medium (Insulin 10 μg ml⁻¹, transferrin 100 μg ml⁻¹, bovine serum albumin 300 μg ml⁻¹, putrescine 16 μg ml⁻¹, thyroxine 400 ng ml⁻¹, tri-iodo-thyronine 300 ng ml⁻¹, progesterone 60 ng ml⁻¹, sodium selenite 40 ng ml⁻¹, 1 mM Glutamax in DMEM, low glucose, pyruvate, Life Technologies). After serum starvation, astrocytes were treated with either pre-clustered rat recombinant EphB1-Fc (1, 5, 10 µg ml⁻¹, R&D Systems) or IL-6 (50 ng ml⁻¹, R&D systems) in Sato’s medium. For all experiments clustered EphB1-Fc was used unless stated otherwise and was referred to as ‘EphB1’ in the text. Before treatment hiPSC-derived astrocytes were cultured for 72 h in absence of BMP4 and LIF and then treated with either human recombinant EphB1-Fc (10 µg ml⁻¹, Gentaur) or IL-6 (50 ng ml⁻¹, R&D systems). Clustering of EphB1-Fc was obtained by incubating EphB1 with a clustering antibody (goat anti-human IgG, Fc fragment specific, 1:10, Jackson ImmunoResearch) for 30 min at RT. At the appropriate time point after the stimulation, cells were further processed for immunocytochemistry, WB or RNA extraction.