Cd11b magnetic bead

Microglia were isolated from post-mortem WM tissue, as described previously [11 (link), 15 (link)]. Briefly, post-mortem tissue that was collected during autopsy was stored in Hibernate-A medium (Invitrogen, Carlsbad, CA, USA) at 4 °C until further processing. Within 24 h, the tissue was homogenized for 5 min in Hibernate-A medium supplemented with DNAseI (10 mg/ml; Roche, Basel, Switzerland), using a tissue homogenizer (VWR, Radnor, PA, USA). Next, undiluted Percoll (density of 1.13 g/ml; GE Healthcare, Little Chalfont, UK) was added to form a single gradient for density centrifugation and the interlayer was collected for magnetic activated cell sorting (MACS; Miltenyi, Bergisch Gladbach, Germany) using CD11b magnetic beads (catalogue number #130–049-601, Miltenyi Biotech). Viable cells were counted using a hemocytometer (Optic Labor, Friedrichshof, Germany) or eFluor™ 506. Cells were then collected in beads buffer (0.5% BSA + 2 mM EDTA in PBS, pH 7.6) for flow cytometry analysis. Using this protocol, about 95% of viable cells were identified as myeloid cells (Supplementary Fig. 1). For CyTOF analysis, CD11b⁺ cells were incubated for 11 min in fixation/stabilization buffer (Smart Tube Inc., San Carlos, CA, USA) and stored in − 80 °C.

Primary neuron cultures were generated from postnatal day 4–10 WT, Nf1^+/neo or Nf1^+/1809 mice. Hippocampi were dissected in Hibernate-A (Gibco) and primary hippocampal neurons were established after papain dissociation, following the manufacturer’s instructions (Worthington). Hippocampal neurons were grown for 7 days prior to analyses. Retinae were dissected in Hibernate-A (Gibco), dissociated in papain (Worthington) and ovomucoid inhibitor (Worthington) before being filtered with CD11b magnetic beads (Miltenyi Biotech) to deplete microglia. The remaining RGCs were plated on poly-D-lysine (Sigma)-coated plates and incubated in neurobasal media supplemented with N2, T3, transferrin, BSA, progesterone, putrescine, sodium selenite, l-glutamine, insulin, N-acetyl cysteine, and forskolin. RGC neurons were grown for 4 days prior to analyses. DRG tissues were isolated in HBSS (Gibco), dissociated in papain (Worthington biochemical) and collagenase type I (STEMCELL Technologies), prior to being strained (70 µm), plated in fibronectin (Fisher)-coated plates, and incubated in 10% fetal bovine serum in DMEM (Gibco). DRG neurons were grown for 7 days.

Adult microglia isolation was performed using MACS, as previously described [47 (link)]. Briefly, mice were anesthetized with avertin and transcardially thoroughly perfused with PBS to remove circulating blood cells in the CNS. Each dissected brain was chilled on ice and then minced in enzymatic digestion buffer containing 0.2% Collagenase Type 3 (Worthington, LS004182) and 3 U/mL Dispase (Worthington, LS02104). Minced brain tissue was then incubated at 37°C for 45 min. The enzymatic digestion was stopped with inactivation buffer containing 2.5 mM EDTA (Thermofisher, 15575020) and 1% fetal bovine serum (Invitrogen, 10082147). The digested brain tissue was then triturated in a serological pipette several times before passing through a 70-μm filter. The homogenate was then depleted of myelin using myelin removal beads (Miltenyi Biotec, 130-096-733) and magnetic LD column (Miltenyi Biotec, 130-042-901). The elute was enriched for microglia with CD11b magnetic beads (Miltenyi Biotec, 130-049-601) and MS column (Miltenyi Biotec, 130-042-201).