Hibernate a medium

Subcortical white matter samples in a range of 5–10 g/donor were stored at 4 °C in Hibernate A medium (Brain Bits LLC, Springfield, IL, USA) until workup. Before workup, a small tissue fragment was snap-frozen in liquid nitrogen and stored at −80 °C for immunohistochemistry. The remaining tissue was mechanically dissociated, followed by enzymatic dissociation, as we described previously^{14 (link)}. After lysis of erythrocytes, mononuclear cells were separated from the suspension by Percoll gradient centrifugation. PBMCs from deceased brain donors, acquired by puncture of either the heart or the iliac artery for collection of 5 ml blood, and from anonymous blood donors (buffy coats; Sanquin Blood Supply Foundation, Amsterdam, The Netherlands) were isolated using standard density gradient centrifugation.

Ad libitum fed Pomc-eGFP mice were sacrificed by cervical dislocation, and tissue from the medial basal hypothalamus (MBH) region was micro-dissected into Hibernate A medium without calcium and magnesium (BrainBits, Springfield, IL, USA). The tissue was then digested with Papain (20 U/ml, Worthington, Lakewood, NJ, USA) for 30 min at 37 °C with mild agitation, followed by trituration in Hibernate A medium containing DNase I (0.005%, Worthington). The cell suspension was then filtered through a 40 μm cell strainer into a new Falcon tube.
Fluorescence-activating cell sorting was performed using an Influx Cell Sorter (BD Biosciences, San Jose, CA, USA) utilizing a protocol that was previously used for single-cell isolation [31] (link), [32] (link). The cell gating was set according to cell size (FSC), cell granularity (SSC), FSC pulse-width for singlets, fluorescence at 488 nm/532 nm for GFP and 647/670 nm for nuclear stain with DraQ5 (Biostatus, Shepshed, Leicester, UK). Cells were sorted direct into a 96 well plate containing lysis buffer with RNase inhibitor.

Mouse pups between postnatal days 0–2 (P0–2) were used for the isolation of primary hippocampal neurons. The hippocampus was removed and placed in cold Hibernate-A medium (BrainBits LLC; Springfield, IL). Hippocampi were then transferred to a digestion medium containing Hibernate-A-CaCl₂ (BrainBits LCC), papain, L-Cysteine, and EDTA for 15 min at 37 °C with occasional gentle shaking. The hippocampi were gently washed in a digestion inhibitor solution and subsequently triturated. The cells were plated in a plating medium at either a density of 650,000 or 250,000 cells per well in a 12-well culture dish (for protein and immunocytochemical analysis, respectively) and the medium was replaced with NbActiv4 (BrainBits LCC) + FdU mitotic inhibitor the following day. PFF was added to the primary cultures between 7 and 10 days in vitro (DIV) at a concentration of 1 μg/ml for immunocytochemical analysis, and 4 μg/ml for western blot analysis. For uptake analysis, PFFs were removed following 2 h of incubation, the cells washed in fresh medium, then replaced with fresh medium. The uptake time points started at time 0 h when PFF was washed from the culture (i.e., 2 h post treatment). For immunocytochemical analysis, 1 μg/ml of PFF was added and the neurons were allowed to continue in culture for up to 14 days.