Hibernate a medium

A sterile working field was set up for the sectioning procedure. This is to avoid any contamination of the tissue being processed for the sectioning. All tools required for this procedure were sterilized following clinical protocols and placed within reach of the experimenter to prevent delays. The sections were prepared using a vibratome (VT1200; Leica Biosystems). The sectioning chamber was filled with ice-cold preparation medium containing Hibernate-A Medium (Lot No. 1994548; Gibco) supplemented with 13 mM d-glucose (Lot No. SLBX3638; Sigma-Aldrich), 30 mM N-methyl-D-GLucamin (M2004; Sigma-Aldrich), and 1 mM GlutaMAX (Lot No. 1978435; Gibco), saturated with carbogen (95% O₂ and 5% CO₂) for 10 min before the sectioning of resected tissue. Millicell inserts (No. PICM03050; Millipore) were placed in each well of a six-well culture dish with 1 ml of growth medium containing Neurobasal l-Glutamine (Lot No. 1984948; Gibco) supplemented with 2% serum-free B-27 (Lot No. 175040001; Gibco), 2% Anti-Anti (Lot No. 15240-062; Gibco), 13 mM d-glucose (Lot No. RNBG7039; Sigma-Aldrich), 1 mM MgSO₄ (M3409; Sigma-Aldrich), 15 mM Hepes (H0887; Sigma-Aldrich), and 2 mM GlutaMAX (Lot No. 1978435; Gibco).

Mice were anaesthetized and transcardially perfused with ice-cold saline. Injured or normal spinal cord segments (8 mm) centered on the lesion site were immediately extracted and maintained in an ice-cold Hibernate-A medium (Gibco). Single-cell suspensions were obtained by using a Neural Tissue Dissociation Kit (P) (Miltenyi Biotec) according to the manufacturer’s protocol. Briefly, the segments were cut into small pieces and then incubated in 50 μL Enzyme P diluted in 1.95 mL Buffer X for 30 min at 37 °C with gentle shaking (100 r.p.m). Enzyme A (10 μL) diluted in 20 μL of Buffer X was next added to the digestive system. Following incubation for 10 min, single-cell suspensions were generated by pipette blowing. The suspensions were then strained using a cell strainer (70 μm) and centrifuged. Cell pellets were resuspended in 33% Percoll (GE Healthcare) layered on 70% Percoll and centrifuged for 15 min at 1800 r.p.m. Cells were obtained from the 30–70% interface and washed with Hank’s Balanced Salt Solution twice. All steps were conducted at 4 °C except digestion.
For ActD based dissociation, ActD (Sigma-Aldrich) was added across all steps during the dissociation at the final concentration of 45 μM at 37 °C for digestion or 3 μM at 4 °C for trituration and cell sorting [26 (link)].

The motility of mouse microglia was determined using mixed cortical cultures, established and cultivated as stated above. A day prior to imaging, the medium was changed to Hibernate A medium (Gibco) in order to maintain a physiological pH value during imaging. Prior to imaging, different wells of cells from each individual pup were treated with either 8 nM MIF, 1.6 nM CXCL4L1, or both (pre-incubated for 30 min on ice to allow for complex formation). A control group was treated with 20 mM sodium phosphate buffer, pH 7.4. Cell motility was monitored by time-lapse imaging for 15 h at 37 °C with recordings every 5 min, using a Leica DMi8 inverted Life Cell Imaging System using the FITC channel for visualizing the GFP-positive cells. Images were imported as stacks to ImageJ software version 1.51n and analyzed with the manual tracking and Chemotaxis and Migration Tool Plugin for ImageJ from Ibidi. In order to quantify microglial motility from the time-lapse videos, 20–25 GFP-positive microglia per treatment group were randomly selected and manually tracked throughout all frames. Cells that died or moved out of the frame were excluded from the analysis. Accumulated distance of each tracked microglia was calculated with Chemotaxis and Migration Tool (Ibidi).

Human postmortem brain and leptomeninges were collected by the New York Brain Bank at Columbia University using an established tissue banking pipeline [37 (link)]. After careful dissection of regions of interest, tissues were stored in Hibernate-A medium (Gibco cat. #A1237501) containing B27 serum-free supplement (Gibco cat #17504044) and stored at 4C until ready for further processing.

Brain was dissected in ice-cold Hank’s balanced salt solution (HBSS) supplemented with sucrose [37 (link)]. A piece of the frontal cortex, hippocampi, and olfactory bulbs were stored in Hibernate-A medium (Gibco, Paisley, UK; A12475–01) during transport at room temperature [21 (link),22 (link)]. Tissues were processed within 2 to 4 h after euthanasia.

To obtain the midfrontal gyrus (BA 9/46) a 1.5 cm × 1.5 cm thick section is cut off from the first cerebral slab anterior to the caudate. After weighing, the tissue is placed in ice-cold transportation medium (Hibernate-A medium (Gibco, A1247501) containing 1% B27 serum-free supplement (Gibco, 17504044) and 1% GlutaMax (Gibco, 35050061)) and shipped overnight at 4 °C with priority shipping. Brain specimens were distributed for this project from autopsies taking place Sunday morning to Thursday. Only autopsies for which the post mortem delay was less than 12 h were included in this study.

Primary hippocampal neuron cultures were prepared from mice by isolating hippocampi from P0-P1 pups of both sexes under a dissection microscope. For mixed cultures, hippocampi were stored in Hibernate-A medium (Gibco) supplemented with 2% B-27 (Gibco) for 1–2 days at 4°C until the second litter was born. Then, hippocampi were incubated with papain (Worthington) at 37°C for 18 min, dissociated by gentle trituration, and plated onto 18 mm glass coverslips treated with poly-L-lysine (Sigma). Electroporations were performed immediately before plating neurons using a Nucleofector 2b Device (Lonza) and the Mouse Neuron Nucleofector Kit (Lonza), following the manufacturer’s instructions. Neurons were maintained in Neurobasal A medium supplemented with 2% B-27% and 1% GlutaMAX (Gibco) in an incubator at 37°C and 5% CO₂. After 5 days in culture, 5 μM cytosine arabinoside (Sigma) was added to inhibit glial division. Subsequently, medium was half exchanged every 3–4 days. For PA-Rac1 experiments, cultures were wrapped in foil to minimize background activity due to ambient light.