Single cell suspensions of all brain regions, i.e., all regions except spinal cord, sympathetic and enteric nervous system as well as dorsal root ganglia, were prepared as described previously (Hochgerner et al., 2018). Briefly, mice were sacrificed with an overdose of isoflurane, followed by transcardial perfusion with artificial cerebrospinal fluid (aCSF, in mM: 87 NaCl, 2.5 KCl, 1.25 NaH2PO4, 26 NaHCO3, 75 sucrose, 20 glucose, 1 CaCl2, 7 MgSO4). The brain was removed, 300μm vibratome sections collected and the regions of interest microdissected under a stereo microscope with a cooled platform. The pieces were dissociated using the Worthington Papain kit, with 25-35 min enzymatic digestion, as needed, followed by manual trituration using fire polished Pasteur pipettes and filtering through a 30μm aCSF-equilibrated cell strainer (CellTrics, Sysmex). Cells were then pelleted at 200 g, 5 min, supernatant carefully removed, and resuspended in a minimal volume aCSF. After manually counting cell concentration using a Burker chamber, suspensions were further diluted to desired concentrations. For improved cell viability, the composition of aCSF was altered (NMDG-HEPES) for experiments using P60 mice (see Table S1)
