Hanks balanced salt solution with calcium and magnesium

Organotypic cerebellar slice cultures were prepared according to a modified protocol published by Stoppini et al.[40 (link)] Shortly, P9‐P10 pups were anaesthetized, and killed, and the cerebellum was removed and cut into 400‐μm‐thick slices using a McIlwain tissue chopper. OSCs were then dissected manually in ice‐cold dissecting medium and transferred to washing medium for 10 min on ice. Thereafter, OSCs were cultured on cell culture inserts with a pore diameter of 0·4 µm (Millipore) in culture medium at 37°C and 5% CO₂ for 3–5 days and subsequently at 33°C and 5% CO2 for the duration of the experiment. The different media used were as follows: dissecting medium—Hank's Balanced Salt Solution with calcium and magnesium (HBSS), 100 U/ml penicillin, 100 μg/ml streptomycin (all from Thermo Fisher Scientific), 5 mg/ml glucose and 1 mM kynurenic acid (both from Sigma‐Aldrich, Seelze, Germany), pH 7·2‐7·4; washing medium—50% HBSS and 50% Minimum Essential Medium (MEM), supplemented with P/S and 25 mM HEPES (all from Thermo Fisher Scientific); and culture medium—50% MEM supplemented with P/S, 25% HBSS supplemented with P/S, 25% horse serum (Thermo Fisher Scientific), 5 mg/ml glucose and 2 mM glutamine.

The E8 fragments of Laminin 211 (LN221F), laminin 411 (LN411F), and laminin 511 (LN511F) were purchased from Nippi, Inc. (Tokyo, Japan). Fibronectin and platelet-poor plasma-derived bovine serum (PDS) were procured from FUJIFILM WAKO (Osaka, Japan). Collagen type IV was purchased from Nitta gelatin Inc. (Osaka, Japan). Human endothelial serum-free medium (HE-SFM), Essential 8 Flex medium, vitronectin (VTN-N), chemically defined lipid concentrate, 1,1′-dioctadecyl-3,3,3′,3′-tetramethyl-indocarbocyanine perchlorate acetylated low-density lipoprotein (Dil-Ac-LDL), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) solution (1 M, pH 7.0–7.6), B-27, Hanks’ balanced salt solution with calcium and magnesium, and lucifer yellow (LY) were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Endothelial Cell Basal Medium 2 was procured from Lonza (Basel, Switzerland). Cell culture inserts (1.0 µm transparent PET membrane) for a 12-well plate and Matrigel GFR were purchased from Corning, Inc. (Corning, NY, USA). Fetal bovine serum (FBS), FITC-dextran 4000 (FD4), and gelatin were procured from Sigma-Aldrich Corporation (St. Louis, MO, USA). TC Protector was purchased from DS Pharma Biomedical (Osaka, Japan). KnockOut Serum Replacement (KSR) was procured from Invitrogen (Carlsbad, CA, USA). A-83-01 was purchased from Cayman Chemical (Ann Arbor, MI, USA)