Mem eagle medium

HT29/B6 cells were kept on at 37°C in a 5% CO₂ environment until reach confluence. Subsequently, a defined scratch (diameter 100 μm) was introduced to filter-grown HT29/B6 cells and kept with medium with 1% of fetal bovine serum (Gibco) to avoid cell proliferation. Cells were exposed to IL-22 (10 ng/ml) and migration was evaluated by measuring the distance at 24 and 48 h after scratching. To perform invasion assay, Matrigel® was diluted (1 mg/ml), placed 100 μl into upper chamber of 24-well transwell and incubated at 37°C for 4–5 h. Subsequently, 2 × 10⁵ CaCo-2 cells in 100 μl plus 100 μl of media (MEM Eagle Medium, Gibco + 1% penicillin and streptomycin, Corning) without FBS were placed into the transwell chamber with Matrigel® cells were treated with or without IL-22 (100 ng/ml). In transwell lower chamber was added 600 μl of culture media and then, incubated for 24 h. The transwell chamber was removed and cells presented in the lower chamber were washed 2× with PBS⁺, fixed (PFA 2%) at room temperature for 30 min and stained with DAPI (1:2000 for 30 min). Number of colonies were counted and analyzed by confocal laser scanning microscopy (LSM 780, Carl Zeiss, Jena).

Organotypic hippocampal slices were prepared from 5-7-day-old C57Bl6 pups according to Stoppini et al. 1991 [26 (link)] with slight modifications as described in [13 (link)]. Briefly, the hippocampi were extracted in carbonated low Na cerebrospinal fluid (CSF) (1 mM CaCl₂, 10 mM D-glucose, 4 mM KCl, 5 mM MgCl₂, 26 mM NaHCO₃, 234 mM sucrose and 0.1% phenol red solution) and coronal slices of 400 μm were made using a tissue chopper (Stoelting, #51425). Hippocampal slices with intact dentate gyrus (DG) and cornu ammonis (CA) regions were selected and maintained on air-fluid interface-style Millicell culture inserts, 30 mm diameter, 0.4 μm (Millipore, #PICM0RG50) in 6-well culture plates (ThermoFisher Scientific) with 800 μL of 37°C pre-heated sterile medium (MEM Eagle medium 78.8% (Gibco #11095), 20% heat-inactivated horse serum (Gibco, #16050–122), 1 mM L-glutamine, 1 mM CaCl₂, 2 mM MgSO₄, 170 nM insulin, 0.0012% ascorbic acid, 12.9 mM D-glucose, 5.2 mM NaHCO₃, 300 mM Hepes (Sigma #H3375), pH = 7.28, osmolality adjusted to 317–322). The medium was replaced completely three times per week.