13 mm coverslips

Collagen I matrices with a pore size ranging between 5 and 10 µm were reconstituted and characterized with already published protocols^{8 (link), 19 (link), 31 (link)}. Briefly, 13 mm coverslips (VWR International, Darmstadt, Germany) were cleaned, functionalized with 3-aminopropyltriethoxysilane (Alfa Aesar, Karlsruhe, Germany) to enable subsequent covalent binding of poly(styrene-alt-maleic anhydride) (PSMA; MW 30 000 g/mol, Sigma-Aldrich, Steinheim, Germany) monolayers. On top 3D collagen I matrices were reconstituted as previously described from rat tail collagen solutions (stock concentration 4.1 mg/ml, lot# 3298599, Corning, Amsterdam, Netherlands)^{8 (link)}. Fibrillogenesis took place in phosphate buffer at pH 7.5 with a collagen concentration of 2 mg/ml at 37 °C for 1 h. Collagen matrices were washed three times with PBS and equilibrated with cell culture medium overnight prior to cell seeding.

Astrocyte monocultures were grown on 13 mm coverslips (VWR) for 5–7 days; on the day of injection, the cells were pre‐treated with 20 µM TAT‐GAP19 (Tocris) for 10 min prior to, and during microinjection to reduce dye uptake through Cx43‐containing hemichannels. A single cell was then microinjected with 0.5% lucifer yellow dye (Invitrogen) and 2% neurobiotin dye (Vector) in PBS using the FemtoJet microinjector system (Eppendorf) combined with FemtoTips II (Eppendorf). Injection pressure was set to 100 hPa for 0.1 s injection, constant pressure was kept at 10 hPa, and the needle was left in the injected cell for 5 min at room temperature. Cells on coverslip were then fixed in 4% PFA for 10 min and stained for further microscopic analysis. Images were analyzed in ImageJ software; injection areas were automatically thresholded and overlayed on the DAPI channel, after which the number of nuclei within the injection area were counted.
List of antibodies/dyes for microinjected area staining (all used at 1:500 final dilution):

Anti‐lucifer yellow rabbit – Invitrogen A‐5750

Cy3‐Streptavidin – Vector SA‐1300‐1

Cell culture plates (24-well or 96-well as indicated; BD Biosciences) were prepared two days prior to astrocyte isolation. First, wells were coated with 20 µg/ml poly-l-lysine (PLL) (Sigma, 2636-25MG) in PBS overnight at 37 °C, 5% CO₂. The next day, wells were washed twice with PBS and subsequently coated with 2 µg/ml laminin in PBS (Sigma, L2020) overnight at 37 °C, 5% CO₂. For immunofluorescence, cells were plated onto 13 mm coverslips (VWR) coated with 0.5 mg/ml PLL and 10 µg/ml laminin. For calcium imaging, cells were plated onto glass bottom dishes (MatTek, P35G-1.5-14-C) coated with 0.5 mg/ml PLL and 10 µg/ml laminin. Astrocyte isolation was performed as described above under sterile conditions. ACSA-2 labeled cells were flushed from the MS column with pre-warmed AstroMACS medium (Miltenyi Biotec, 130-117-031) supplemented 50 U/ml penicillin/streptomycin (Sigma, P0781-20ML) and 0.25% l-glutamine (0.5 mM; Thermo Fisher, 25030-024). Neonatal and adult astrocytes were cultured using the same medium composition. Cells were plated at 100,000 cells/24-well or glass dish or 25,000 cells/96-well onto the middle of the well in a droplet of AstroMACS medium and incubated at 37 °C, 5% CO₂ for 1–3 h before filling up the medium. The medium was changed every three days and grown for 7–10 days before use.