Application protocol

Adult neural stem cell isolation, characterization, and culture

A workflow protocol to purify neural stem cells (NSCs) from wildtype mouse brain. This entire experimental setting resulted in highly pure (>95%) and viable NSCs (>90%) in less than 3 h.

First, an optimized automated dissociation protocol is applied, which ensures high viability and epitope integrity of the resulting single cell suspension. Then, NSCs are identified by detection of the exclusion markers CD24, Ter-119, and CD45 and the NSC specific markers GLAST and PlexinB2. Subsequently, purification of NSCs is carried out with the MACSQuant Tyto Sorter.  A neurosphere assay can be performed to verify the viability and functionality of the sorted NSCs. 

 MACSQuant Tyto Sorter is a multi-parameter cell sorting device that uses a micro-chip based sorting technology for sterile and gentle cell isolation. Unlike conventional droplet sorters, cells do not experience high pressures and no charge is applied, ensuring high viability and functionality. 

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.

Subventricular zone dissection

  • Dissecting microscope
  • Laminar flow hood
  • ice bucket 
  • 10 cm and 35 mm diameter sterile petri dish
  • Surgical scissors (Hammacher, 130 mm #HCT7.1)
  • (Sterile) scalpel (B. Braun, # 5518083)
  • micro-spatula, round 140 mm/2 mm (ROTH, #AT19.1)
  • Dumont no. 7 forceps, Dumostar (Fine Science Tools, cat. no. 11297-00)
  • Spring scissors, pointed-pointed, 160 mm (ROTH, #3577.1)
  • Dulbecco’s phosphate-buffered saline (D-PBS) with calcium, magnesium, glucose, and pyruvate. Keep buffer cold (2−8 °C).

Dissociation of SVZ tissue 

  • Neural Tissue Dissociation Kit (T), mouse
  • gentleMACS™ Octo Dissociator with Heaters (# 130-096-427) and gentleMACS C Tubes (# 130-093-237, # 130-096-334)
  • MACS SmartStrainer (70 μm) (# 130‑098‑462) 

Debris removal

  • A pre-cooled (4°C) centrifuge with  swinging bucket rotor is recommended. e. g., Heraeus® Multifuge 4KR by Thermo Fisher® Scientific.
  • Polypropylene round-bottom tubes (5 ml, FACS tubes; BD Biosciences, cat. no. 352063)
  • Debris removal solution
  • NSC Antibody cocktail
  • FcR-Blocking reagent, mouse
  • MACSQuantTyto Running Buffer (#130-107-206, #130-107-207). Degas buffer before use, as air bubbles could block the column. 

MACSQuant Tyto sorting

  • Pre-Separation Filter 20 μm (# 130-101-812)
  • MACSQuant Tyto Cartridges (# 130-106-088)
  • 10 mL Syringe (with Luer-Lock tip)
  • (Optional) Extra long pipet tips
  • Cell sorter, e.g., MACSQuant Tyto (# 130-103-931) for cell sorting

Evaluation and analysis of labeling and sorting performances 

  • Flow cytometer, e.g., MACSQuant Analyzer 10 (# 130-096-343)
     
  • (Optional, for analysis only) Propidium Iodide Solution (# 130-093-233) or 7-AAD for flow cytometric exclusion of dead cells without fixation
     
  • (Optional, for analysis only) Anti-EGF-Receptor antibody for flow cytometric analysis of activated and quiescent NSCs
     

Neurosphere assay

  • MACS® Neuro Medium (# 130-093-570)
  • MACS NeuroBrew®-21 (# 130-093-566) 
  • 20 ng/ml EGF
  • 20 ng/ml FGF-2
  • 200 mM L-Glutamine
  • Penicillin/Streptomycin
  • ultra-low attachment plate
  • Imaging Plate CG 1.5 (24 well)
  • Poly-L-Lysin (0,01%)

Immunocytochemical staining of differentiated Neurospheres

  • 2% Paraformaldehyd (PFA)
  • AutoMACS Running Buffer
  • FcR-Blocking reagent, mouse
  • 0.2% Triton-X-100
  • specific antibodies, e.g. Anti-GLAST (for astrocytes), Anti-MAP2 (for neurons), Anti-O4 (for oligodendrocytes)
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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.