Application protocol

Neural stem cell isolation by cell sorting, using the MACSQuant® Tyto®

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, e.g., ????????

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.

Note: 

  • Identification and sorting of NSCs is possible with SVZ tissue of one mouse.
  • For subsequent neurosphere assay, it is recommended to dissociate SVZ tissue of at least 5 mice.
  • Volumes given below are for SVZ tissue of 5 adult mice in 1980 μL enzyme mix. When working with less than SVZ tissue of x mice, use the same volumes as indicated. When working with higher tissue quantities, scale up all reagent volumes and total volumes accordingly. A maximum SVZ tissue of x mice per tube can be processed.
  • Always use pre-cooled buffers and solutions (4 °C) and perform all steps on ice.

Preparation of cell culture plates

Coat a 24-well culture dish with 0.01% Poly-L-lysine overnight at 37 °C. Wash three times with ddH₂O the following day.

Preparation of medium for cell culture

Prepare the following cell culture medium: MACS Neuro Medium containing 2% MACS NeuroBrew®-21, 1% penicillin/streptomycin and 0.5 mM L-glutamine.

Use the Neural Tissue Dissociation Kit (T) to dissociate brain tissue and prepare a single-cell suspension. Follow the protocol of the kit data sheet.

Download data sheet

Neural Tissue Dissociation Kits

Good to know

The data sheet for the Neural Tissue Dissociation Kits includes a set of tips & hints to improve the quality and yield of the dissociation procedure. Refer to the data sheet appendix if, for example, the yield of viable cells is too low or a cell pellet will not form.

Isolate the GLAST-positive astrocytes from the single-cell suspension using the Anti-GLAST (ACSA-1) MicroBead Kit. Follow the protocol of the kit data sheet.

Download data sheet

Anti-GLAST (ACSA-1) MicroBead Kit, human, mouse, rat

Anti-GLAST (ACSA-1) MicroBead Kit, human, mouse, rat
Mouse forebrain cells before separation
GLAST (ACSA-1)-negative cells
GLAST (ACSA-1)-positive cells

Effective isolation of GLAST+ cells from mouse brain tissue. Mouse brain tissue (P7) was dissociated using the Neural Tissue Dissociation Kit (T), the gentleMACS™ Dissociator, and FcR Blocking Reagent, mouse. Astrocytes were isolated from single-cell suspension using the Anti-GLAST (ACSA-1) MicroBead Kit, a MiniMACS™ Separator and an MS Column. Cells were fluorescently stained with Anti-GLAST (ACSA-1)-APC and analyzed by flow cytometry on the MACSQuant® Analyzer. Cell debris and dead cells were excluded from the analysis based on scatter signals and propidium iodide fluorescence.

Notes:

  • The recommended antibody dilution for labeling cells is 1:10 for up to 1×10⁶ cells/50 μL of PB buffer.
  • Volumes given below are for up to 1×10⁶ nucleated cells. When working with fewer than 1×10⁶ cells, use the same volumes as indicated. When working with higher cell numbers, scale up all reagent volumes and total volumes accordingly (e.g., for 2×10⁶ nucleated cells, use twice the volume of all indicated reagent volumes and total volumes).
  1. Use 100 μL of the GLAST+ fraction for analysis. Optionally, also analyze 100 μl of the negative fraction and 20 μL of the original fraction.
  2. Resuspend up to 1×10⁶ nucleated cells per 45 μL of PB buffer (see "Things to prepare in advance of cell isolation and cell culture").
    ▲ Note: Always use freshly prepared buffer.
  3. Add 5 μL of Anti-GLAST (ACSA-1)-APC, mouse antibodies.
  4. Mix well and incubate for 10 minutes in the refrigerator (2−8 °C) in the dark.
    Note: Higher temperatures and/or longer incubation times may lead to non-specific cell labeling. Working on ice requires increased incubation times.
  5. Wash cells by adding 1 mL of PB buffer and centrifuge at 300×g for 10 minutes. Aspirate supernatant completely.
  6. Resuspend cell pellet in a suitable amount of PB buffer for analysis by flow cytometry, e.g., using the MACSQuant® Analyzer 10.
  1. Plate 5×104 cells in 50 μL of pre-warmed prepared medium as a drop in the middle of each well of a coated 24-well plate (see "Things to prepare in advance of cell isolation and cell culture").
  2. Let the cells settle down for 30 minutes at 37 °C in the incubator.
  3. Carefully add 450 μL of prepared medium to each well.
  4. Maintain the culture by replacing 50% of medium every other day.

Things to prepare in advance

Staining buffer: Prepare a solution containing autoMACS® Running Buffer with FcR Blocking Reagent, mouse  in a ratio of 1:10, e.g., add 1 mL FcR Blocking Reagent to 9 mL autoMACS Running Buffer.
  1. Wash cells 3× with PBS.
  2. Fix cells with 2% PFA for 10 minutes at room temperature.
  3. Wash cells 3× with PBS.
    Note: Fixed cells can be stored in azide-containing buffer at 2–8 °C for up to 1 week.
  4. Add staining buffer (see "Things to prepare in advance") and incubate for 10 minutes at room temperature.
  5. Discard staining buffer.
  6. Add primary antibody in staining buffer to the cells with a final concentration of 1–5 μg/mL and incubate at room temperature in the dark for 10 minutes.
  7. Wash cells 3× with autoMACS Running Buffer.
  8. Add a corresponding secondary antibody in staining buffer to the cells and incubate at room temperature in the dark for 10 minutes.
  9. Wash cells 3× with autoMACS Running Buffer.
    Note: For co-staining with additional antibodies repeat steps 6–9.
  10. Store cells in autoMACS Running Buffer.
  11. Cells are now ready for immunofluorescence microscopy.
    Note: Samples can be stored at 2–8 °C in the dark for up to one week
    Note: When working with cells cultured on coverslips, the coverslips need to be mounted onto slides before imaging.
View details

Successful culture of GLAST-positive astrocytes isolated from neonatal mouse brain tissue. P3 whole mouse brains were dissociated using the Neural Tissue Dissociation Kit (T) and astrocytes were isolated from the single-cell suspension using the Anti-GLAST MicroBead Kit. Astrocytes were cultured in MACS® Neuro Medium, MACS NeuroBrew®-21, 1% P/S, and 0.5 mM L-glutamine on PLL-coated glass coverslips (5×104 cells per well). After 3 days (A) and 6 days (B), cells were fixed and stained with astrocyte-specific antibodies Anti-GLAST (green) and Anti-GFAP (red).

  1. Mich, J.K. et al. (2014) Elife. 2014 May 7;3:e02669. doi: 10.7554/eLife.02669.
  2. Götze et al., Prospective isolation of adult neural stem cells from the mouse subependymal zone, nature protocol, 17 November 2011;

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