Permanox 4 well chamber slides

To investigate migration a protocol for the scratch test/wound healing was used [54 (link), 55 (link)]. In short, Permanox 4 well Chamber slides (Gibco), were used for the procedure. In the bottom of the slides, five vertical reference lines were made with a scalpel. Primary astrocytes, 60,000 cells/mL were then seeded into the poly-D lysine coated dishes. The cells were grown for 24 h—16 of these in FBS-free medium to inhibit cell proliferation during the assay until 80% confluent. A horizontal scratch was made with a 10-µL pipette tip, and the medium was carefully changed to pre-warmed DMEM [56 (link), 57 (link)]. Pictures were taken at t₀, and the test substances were added. These included 25 ng/mL FGF8, collected HTRA1-containing medium in the ratio to FGF8 determined in the activity assay (0.12 µL), and 50 ng/mL MIF. In assays where MIF and HTRA1 were added in combination with the cells, they were pre-incubated for at least 5 min before the addition. After addition of the test substances, the cells were incubated for an additional 24 h. Pictures taken after this time were designated t₂₄. The pictures were taken at the intersections of each reference line with the scratch, thereby ten pictures were taken from each well and it was ensured that t₀ and t₂₄ were captured at the same positions within the wells.

For experiments involving astrocytes, brains of P1–P5 mice were mechanically dissociated and the obtained cells cultivated in DMEM supplemented with 10% fetal bovine serum and penicillin–streptomycin for a minimum of 10 days. The protocol of de Vellis and Cole was followed for purification of astrocytes [53 (link)]. In short, the flasks containing mixed glial cultures were placed on a rotary shaker at 37 °C, 200 rpm, first for 4 h to remove microglia. The medium with microglia was discarded, and new medium was added and the flasks shaken for additional 12–18 h under same conditions. The medium was then collected and centrifuged at 1000 rpm, The flasks were washed with pre- warmed medium once and then medium containing Dulbecco’s Modified Eagle Medium (DMEM) with glutamax containing 10% fetal bovine serum (FBS) and 1× penicillin and streptomycin (Gibco) was added to the astrocytes that adhered to the bottom. Purified astrocytes were removed using trypsin and re-plated in Permanox 4 well Chamber slides (Gibco) for further processing.