Neuron isolation kit

VSV (Indiana strain) and VSV expressing GFP (VSV-GFP) were provided by Glen Barber. For infections, 1 × 10⁷ pfu of VSV in 30 μl of endotoxin-free PBS was administered in drops (~5 ml/drop) to isofluorane-anesthetized 8–12 week-old mice, with PBS-only as control. Thereafter, mice were monitored daily for disease symptoms and mortality. In some experiment, mice were sacrificed 24 h after virus infection, the brain were collected for RNA extraction or for preparation of neural cells by using Neural Tissue Dissociation Kit (Miltenyi Biotech). The isolated neural cells were subjected for flow cytometric analysis or for FACS sorting of microglia (CD11b⁺), astrocyte (GLAST-1⁺), and neuron (CD11b⁻CD31⁻GLAST1⁻O4⁻) by using the Neuron isolation kit from Miltenyi Biotec.

After 7 days of differentiation, the cells were dissociated into single-cell suspensions using Accutase. Neurons were enriched using the Neuron Isolation Kit (Miltenyi Biotec Inc., Auburn, CA, USA) by depletion of non-neuronal cells using the MACS technique, according to the manufacturer’s recommendations. Briefly, non-neuronal cells were indirectly magnetically labelled with biotin-conjugated antibodies and Anti-Biotin MicroBeads (Miltenyi Biotec Inc.). Subsequently, the magnetically labelled non-neuronal cells were retained within a MACS Column, which was placed in the magnetic field of a MACS Separator (Miltenyi Biotec Inc.), while the unlabelled neurons ran through. Furthermore, astrocytes and oligodendrocytes were respectively enriched using the anti-GLAST (ACSA-1) MicroBead Kit and anti-O4 MicroBeads (Miltenyi Biotec Inc.) according to the manufacturer’s instructions. After MACS enrichment, neurons, astrocytes and oligodendrocytes were counted using flow cytometry and immunofluorescence staining for analysis of purity and identity. Subsequently, neurons, astrocytes and oligodendrocytes were separately plated onto PLL-coated 24-well plates (5 × 10⁴ cells per well) and treated with CHI mouse serum for 45 min at 37 °C as described above.

Whole brains from adult female 10- to 15-wk-old mice were dissociated using the Adult Brain Dissociation Kit and Gentle Macs Octo Dissociator with Heaters (Miltenyi) according to the manufacturer’s protocol. Non-neuronal cells were removed using the Neuron Isolation Kit (Miltenyi), which contains biotinylated antibodies against microglia, oligodendrocytes, and astrocytes, conjugated to magnetic microbeads that are removed by magnetic-activated cell sorting (MACS). The flow-through containing highly enriched neuronal cells was collected. The purity of neuronal isolates was assessed by flow cytometry using a FACS Canto II (BD Biosciences) according to the manufacturer’s instructions. In brief, living cells were gated for single cells only (side scatter and forward scatter), and the samples were stained for cell populations using specific antibodies for microglia/macrophages (CD11b PeCy7; Invitrogen), oligodendrocytes (O4 APC; Miltenyi), endothelial cells (CD31 PEVio770; Miltenyi), thrombocytes/immune cells (CD45 FITC; Biolegend), and astrocytes (astrocyte cell surface antigen-2 [ACSA-2] PE, Miltenyi; Table 1). Neuronal purity of 90–96% was achieved.

Isolation of neuron and glia enriched population of cells was performed using the Neuron isolation kit (Miltenyi Biotec) as per the manufacturer’s instructions. Isolated cells were collected in Trizol for RNA extraction and stored at -80° C until further analysis.