Application protocol

Isolation and cultivation of neurons from neonatal mouse brain

This application protocol describes the generation of highly purified and viable neurons from neonatal mouse brain tissue. Brain tissue from mice younger than P8 is dissociated into a single-cell suspension and neurons are then isolated using an indirect magnetic labeling system that depletes non-neuronal cells like astrocytes, oligodendrocytes, microglia, endothelial cells, and fibroblasts. The cell number and composition of the resulting highly pure neuronal cell fraction vary according to mouse age and brain region. This isolation protocol has been tested with CD-1® mice aged from embryonic day 18 (E18) to adult.

Materials

The following is a listing of reagents, instruments, and consumables needed for each step of this protocol. These products are for research use only.

General reagent and instrument requirements

  • DPBS/BSA buffer: Prepare a solution containing Dulbecco’s phosphate-buffered saline (DPBS) with Ca2+ and Mg2+ and 0.5% bovine serum albumin (BSA) by diluting MACS® BSA Stock Solution (# 130‑091‑376) 1:20 with DPBS. Keep buffer cold (2−8 °C). Degas buffer before use, as air bubbles could block the column.
    ▲ Note: Always use freshly prepared buffer. Do not use autoMACS® Running Buffer or MACSQuant® Running Buffer as they contain a small amount of sodium azide that could affect the results.Pre-Separation Filters (70 μm) (# 130-095-823)

For brain tissue dissociation

  • Neural Tissue Dissociation Kit – Postnatal Neurons (# 130-094-802)
  • gentleMACS™ Dissociator (# 130-093-235), gentleMACS Octo Dissociator (# 130-095-937), or
    gentleMACS Octo Dissociator with Heaters (# 130-096-427)
  • gentleMACS C Tubes (# 130-093-237, # 130-096-334)
  • MACS SmartStrainers (70 μm) (# 130-098-462)
  • 35 mm diameter sterile petri dish
  • Sterile glass Pasteur pipettes
  • 50 mL tubes
  • Centrifuge with swinging bucket rotor
  • (Optional) Beta-mercaptoethanol, 50 nM
  • (Optional) ART® 1000 REACH™ pipet tips (Molecular BioProducts, Inc.) for removal of dissociated material from the closed C Tubes.

For cell isolation and flow cytometry analysis

  • Neuron Isolation Kit, mouse (# 130-115-389; small size #130-115-390)
  • MACSmix™ Tube Rotator (# 130-090-753) in combination with an incubator oven at 37 °C
  • MACS Columns and MACS Separators: neurons can be enriched by depletion using LS Columns. Depletion can also be performed by using the autoMACS Pro Separator.
ColumnMax. number of labeled cellsMax. number of total cellsSelector
LS2×10⁷ 4×10⁷MidiMACS™, QuadroMACS™
autoMACS®5×10⁷1×108autoMACS Pro
  • Red Blood Cell Lysis Solution (10×) (# 130-094-183)
  • Fluorochrome-conjugated antibodies for flow cytometry analysis, e.g., Anti-Biotin antibodies conjugated to PE or APC, or Anti-ACSA-2-PE (#130-116-243), Anti-O4-PE (#130-109-199), CD11b-FITC (# 130-110-837). Learn more about our antibodies and dyes.
  • (Optional) Propidium Iodide Solution (# 130-093-233) or 7-AAD for flow cytometry exclusion of dead cells
  • MACSQuant Analyzer 10 (# 130-096-343)

For cell culture

  • Double-distilled water (ddH₂O)
  • Imaging Plate CG 1.5 (24 well) (# 130-098-263)
  • MACS Neuro Medium (# 130-093-570)
  • MACS NeuroBrew®-21 (# 130-093-566)
  • L-glutamine
  • Poly-L-lysine (0.01%)
  • Penicillin/streptomycin

For immunocytochemical staining of cultured cells

  • Primary antibody of choice, e.g., Anti-ACSA-2 pure, mouse (# 130-099-138), Anti-GLAST (ACSA-1) pure, human, mouse, rat (# 130-095-822), Anti-O4 pure, human, mouse, rat (# 130-115-810), Anti‑PSA‑NCAM pure, human, mouse, rat (# 130-115-809), CD11b pure, human and mouse (# 130-115-811), CD68 pure, mouse (# 130-115-808), or CD171 (L1CAM) pure, mouse (# 130-115-812)
  • A corresponding secondary antibody, e.g., Anti-rat IgG2b, Anti-mouse IgG2a, Anti-mouse IgM, Anti-rat IgG2bκ, Anti-rat IgG2a
    Note: Store antibodies in aliquots at –20 °C. To avoid repeated freeze-thaw cycles prepare working aliquots.
  • Staining buffer: Prepare a solution containing autoMACS Running Buffer (# 130-091-221) with FcR Blocking Reagent, mouse (# 130-092-575) (1:10).
  • Phosphate-buffered saline (PBS)
  • autoMACS Running Buffer (# 130-091-221)
  • (Optional) 0.2% TRITON™ X-100 in PBS
  • Distilled water
  • 2% paraformaldehyde (PFA) for fixation
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Protocol

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This protocol includes detailed instructions for preparing necessary reagents and materials and then carrying out each step.