Application protocol

Isolation and cultivation of CD171 (L1CAM)-positive neurons from neonatal mouse brain

The CD171 (L1CAM) (L1 cell adhesion molecule) antigen is expressed by tetanus-toxin positive neurons, endothelial cells, certain epithelial cells, reticular fibroblasts, and several malignant tumors, including colon and breast carcinomas, colon melanoma, and tumor cells of neuronal and mesothelial origin. CD171 plays a vital role in cell adhesion and signal transduction, and has been demonstrated to play a role in augmenting tumor growth. It is involved in the development of the nervous system and regulates processes such as neuron–neuron adhesion, myelination, axonal guidance, and neuronal migration. CD171 (L1CAM) MicroBeads are designed for the separation of neurons based on the expression of CD171 (L1CAM), and especially optimized for use with dissociated postnatal CD-1® mouse brain tissue derived from animals younger than postnatal day eight (P8). Up to 99.5% purity can be achieved with P7 cerebellum tissue because the expression of CD171 is found almost exclusively on neurons. Purity decreases with age: P9 up to 80%; P12 up to 64%. For other brain regions, glial cells may need to be depleted if high purities are required. The CD171 (L1CAM) epitope shows papain sensitivity. Therefore, the Neural Tissue Dissociation Kit (T) is recommended to generate a single-cell suspension.

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.

For brain tissue dissociation

  • Neural Tissue Dissociation Kit (T) (# 130-093-231)
  • Hanks´ Balanced Salt Solution (HBSS) without Ca2+ and Mg2+ (Sigma-Aldrich # 55021C)
  • HBSS with Ca2+ and Mg2+ (Sigma-Aldrich # 55037C)
  •  (Optional) Beta-mercaptoethanol, 50 mM
  •  50 mL tubes
  • MACS® SmartStrainer (70 μm) (# 130-098-462) for 50 mL tube
  • MACSmix™ Tube Rotator (# 130-090-753) in combination with an incubation oven at 37 °C
  • (Optional) gentleMACS™ Dissociator (# 130-093-235), gentleMACS Octo Dissociator (# 130-095-937), or gentleMACS Octo Dissociator with Heaters (# 130-096-427)
  • C Tubes (# 130-093-237, # 130-096-334)
  • (Optional) ART® 1000 REACH™ pipet tips (Molecular BioProducts, Inc.) for removal of dissociated material from the closed C Tubes.
  •  (Optional) Myelin Removal Beads II, human, mouse, rat (# 130-096-433, # 130-096-733).

For cell isolation and flow cytometry analysis

  • CD171 (L1CAM) MicroBead Kit, mouse (# 130-101-548) or CD171 (L1CAM) MicroBead Kit, mouse - small (# 130-101-549)
  • Pre-Separation Filters (70 μm) (# 130-095-823)
  • 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: BSA can be replaced by other proteins such as mouse serum albumin, mouse serum, or fetal bovine serum (FBS).
  • MACS Columns and MACS Separators: CD171 (L1CAM)+ cells can be enriched using MS or LS Columns or depleted with the use of LD Columns. Positive selection or depletion can also be performed by using the autoMACS® Pro Separator. 
ColumnMax. number of labeled cellsMax. number of total cellsSeparator
Positive selection
MS1×10⁷2×10⁷MiniMACS™, OctoMACS™,
VarioMACS, SuperMACS II
LS2×10⁷4×10⁷MidiMACS™, QuadroMACS,
VarioMACS, SuperMACS II
Depletion
LD1.5×10⁷3×10⁷MidiMACS, QuadroMACS,
VarioMACS, SuperMACS II
Positive selection or depletion
autoMACS5×10⁷ 1×108autoMACS Pro
Note: Column adapters are required to insert certain columns into the VarioMACS™ or SuperMACS™ II Separators. For details refer to the respective MACS Separator data sheet.
  • Labeling Check Reagent-APC (# 130-098-892) to stain labeled cells for flow cytometry analysis.
    Note: The use of CD171 antibodies, clone 555, is not recommended for analysis of cells that are labeled with CD171 (L1CAM) MicroBeads. Learn more about our antibodies and dyes.
  • Propidium Iodide Solution (# 130-093-233) or 7-AAD for flow cytometry exclusion of dead cell
  • 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.