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
Matching products:

Protocol


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

Preparation of buffer for tissue dissociation, neuron isolation and flow cytometry

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 1:20 with DPBS. Keep buffer cold (2–8 °C). Prepare fresh on the day of use and degas the buffer, as air bubbles could block the column.

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

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

Dissociate mouse neonatal brain using the Neural Tissue Dissociation Kit – Postnatal Neurons. Follow the protocol of the kit data sheet.

Download data sheet

Neural Tissue Dissociation Kit – Postnatal Neurons

Isolate neurons from the single-cell suspension using the Neuron Isolation Kit, mouse. Follow the protocol of the kit data sheet.

Download data sheet

Neuron Isolation Kit, mouse

Notes:

  • The recommended antibody dilution for labeling of cells is 1:10 for up to 1×10⁶ cells/50 μL of DPBS/BSA 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. Analyze 100 μL of the positive and 100 µL of the negative fractions. Optionally, also analyze 20 μL of the original fraction.
  2. Centrifuge at 300×g for 5 minutes. Aspirate supernatant completely.
  3. Resuspend up to 1×10⁶ nucleated cells per 45 μL of DPBS/BSA buffer (see "Things to prepare in advance of tissue dissociation and cell culture").
  4. Add 5 μL of Anti-Biotin-PE.
  5. Mix well and incubate for 10 minutes in the dark in the refrigerator (2−8 °C).
    Note: Higher temperatures and/or longer incubation times may lead to non-specific cell labeling. Working on ice requires increased incubation times.
  6. Wash cells by adding 1 mL of DPBS/BSA buffer and centrifuge at 300×g for 10 minutes. Aspirate supernatant completely.
  7. Resuspend cell pellet in a suitable amount of DPBS/BSA buffer for analysis by flow cytometry, e.g., using the MACSQuant® Analyzer 10.
  1. Plate 1×10⁵ cells in 50 μL of prepared medium as a drop in the middle of each well of a coated 24-well plate (see "Things to prepare in advance of tissue dissociation and cell culture").
  2. Let cells settle for 30 minutes at 37 °C in the incubator.
  3. Carefully add 450 μL of prepared medium to each well.
  4. Take off the medium to remove non-attached and dead cells.
  5. Add 500 μL of prepared medium.
  6. 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 (1:10).

Immunocytochemical staining of cultured neurons

  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: When working with CD68 antibodies, add 0.2% TRITON X-100 in PBS, incubate for 10 minutes at room temperature, and wash cells 3× with autoMACS® Running Buffer. Do not treat cells with TRITON X-100 before staining with Anti-ACSA-2, Anti-O4, or Anti-PSA-NCAM antibodies.
    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 pure antibody of choice in staining buffer to the cells and incubate in the dark. For incubation temperature and time as well as recommended antibody concentration, refer to the table below.
  7. Wash cells 3× with autoMACS Running Buffer.
  8. Add a corresponding secondary antibody in staining buffer to the cells and incubate in the dark. For incubation temperature and time as well as recommended antibody concentration, refer to the table below.
  9. Wash cells 3× with autoMACS Running Buffer.
    Note: For co-staining with additional antibodies repeat step 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.
Primary AntibodySecondary Antibody
AntibodyIncubation temperatureIncubation timeRecommended antibody concentration Secondary antibodyIncubation temperatureIncubation time
Anti-ACSA-2Room temperature10 minutes1–5 µg/mLAnti-rat IgG2bRoom temperature10 minutes
Anti-GLAST (ACSA-1)Room temperature10 minutes 1–5 µg/mLAnti-mouse IgG2aRoom temperature10 minutes
Anti-O4Room temperature10 minutes 5–10 µg/mLAnti-mouse IgMRoom temperature10 minutes
Anti-PSA-NCAMRoom temperature10 minutes  5–10 µg/mLAnti-mouse IgMRoom temperature10 minutes
CD11bRoom temperature10 minutes  5–10 µg/mLAnti-rat IgG2bκRoom temperature10 minutes
CD682–8 °COvernight  5–10 µg/mLAnti-rat IgG2aRoom temperature1 hour
CD171 (L1CAM)2–8 °COvernight  5–10 µg/mLAnti-rat IgG2aRoom temperature1 hour
View details

High-purity neuron population in culture. P1 mouse cerebral hemispheres were dissociated using the Neural Tissue Dissociation Kit – Postnatal Neurons. Subsequently, neuronal cells were isolated using the Neuron Isolation Kit, mouse. Neurons were cultured in MACS® Neuro Medium, MACS NeuroBrew®-21, 1% P/S, and 0.5 mM L-glutamine on PLL-coated glass coverslips (1×105 cells per well of a 24-well plate). After 2 days (A) and 6 days (B), in vitro cells were fixed and stained with the neuron-specific antibody Anti-MAP2 (red) and the astrocyte-specific antibody Anti-GLAST (green).

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