Application protocol

Isolation and cultivation of O4-positive oligodendrocytes from neonatal mouse brain

This application protocol describes the isolation of highly purified and viable oligodendrocytes from neonatal mouse brain tissue. Brain tissue from mice younger than P8 is dissociated into a single-cell suspension and Anti-O4 MicroBeads are used to isolate oligodendrocytes. The O4 antigen, a sulfatide of the glycosphingolipid class is a marker for oligodendrocytes. During oligodendrocyte development, O4 expression begins on late oligodendrocyte progenitors that are A2B5-positive. While A2B5 expression disappears, O4 continues to be expressed. O4 expression is also found in Schwann cells. The isolation of O4+ cells leads to highest purities if P3–P7 (postnatal day 3–7) rodents are used. Mouse brain tissue derived from P3–P7 CD-1® mice contains approximately 5–10% O4+ cells.

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 (P) (#130-092-628) or Neural Tissue Dissociation Kit (T) (# 130-093-231)
  • Hanks' Balanced Salt Solution (HBSS) without Ca2+ and Mg2+
  • HBSS with Ca2+ and Mg2+
  • 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)
  • (Optional) ART® 1000 REACH™ pipet tips (Molecular BioProducts, Inc.) for removal of dissociated material from the closed C Tubes.
  • (Optional) Beta-mercaptoethanol, 50 mM
  • MACS® SmartStrainers (70 µm) (# 130-090-753) in combination with an incubator at 37 °C
  • MACSmix™ Tube Rotator (#130-098-753)
  • 50 mL tubes

For cell isolation and flow cytometry analysis

  • Anti-O4 MicroBeads, human, mouse, rat (#130-094-543; small size 130-096-670)
  • FcR Blocking Reagent, mouse (# 130-092-575) or FcR Blocking Reagent, human (#130-059-901)
  • Pre-Separation Filters (70 µm) (#130-095-823)
  • Fluorochrome-conjugated Anti-O4 antibodies for flow cytometry analysis, e.g., Anti-O4-PE (# 130-095-887) or Anti-O4-APC (# 130-095-891). Learn more about our antibodies and dyes.
  • Propidium Iodide Solution (#130-093-233) or 7-AAD for flow cytometric exclusion of dead cells.
  • (Optional) MACSQuant® Analyzer 10 (# 130-096-343)
  • PB buffer: Prepare a solution containing phosphate-buffered saline (PBS), pH 7.2 and 0.5 % bovine serum albumin (BSA) by diluting MACS® BSA Stock Solution (# 130-091-376) 1:20 with PBS. 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 or rat serum albumin, mouse serum, or fetal bovine serum (FBS).
  • MACS Columns and MACS Separators: O4+ cells can be enriched using LS or MS Columns. Positive selection can also be performed using the autoMACS® Pro Separator or the MultiMACS™ Cell24 Separator.
ColumnMax. number of labeled cellsMax. number of total cellsSeparator
Positive selection
MS1×1072×107MiniMACS™, OctoMACS™
LS2×1074×107MidiMACS™, QuadroMACS™
autoMACS5×1071×108autoMACS Pro
Multi-242×1074×107MultiMACS Cell24

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)
  • 200 mM L-glutamine
  • Poly-L-lysine (0.01%)
  • Penicillin/streptomycin
  • Human PDGF-AA (# 130-093-977)
  • Human FGF-2 (# 130-093-837) 

For immunocytochemical staining of cultured cells

  • Anti-O4 pure, human, mouse, rat (# 130-115-810) and anti-mouse IgM secondary antibody
  • Staining buffer: Prepare a solution containing autoMACS Running Buffer (# 130-091-221) with FcR Blocking Reagent, mouse (# 130-092-575) in a ratio of 1:10, e.g., add 1 mL FcR Blocking Reagent to 9 mL autoMACS Running Buffer.
  • Phosphate-buffered saline (PBS)
  • autoMACS Running Buffer (# 130-091-221)
  • 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.

Preparation of buffer for cell isolation and flow cytometry

PB Buffer: Prepare a solution containing phosphate-buffered saline (PBS), pH 7.2, and 0.5 % bovine serum albumin (BSA) by diluting MACS® BSA Stock Solution 1:20 with PBS. Keep buffer cold (2−8 °C). Prepare fresh on the day of use and degas the buffer, 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).

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, 10 ng/mL Human PDGF-AA, and 10 ng/mL Human FGF-2.

Dissociate mouse neonatal brain using the Neural Tissue Dissociation Kit (P) or Neural Tissue Dissociation Kit (T). Follow the protocol of the kit data sheet.

Download data sheet

Neural Tissue Dissociation Kits

Isolate O4-positive oligodendrocytes using the Anti-O4 MicroBeads, human, mouse, rat. Follow the protocol of the kit data sheet.

Download data sheet

Anti-O4 MicroBeads, human, mouse, rat

Notes:

  • The recommended antibody dilution for labeling cells is 1:11 for up to 1×10⁷ cells/100 μL of PB buffer.
  • Volumes given below are for up to 1×10⁷ nucleated cells. When working with fewer than 1×10⁷ 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 2×10⁷ 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 include 20 µL of the original fraction in the analysis.
  2. Resuspend up to 1×10⁷ nucleated cells per 100 μL of PB buffer (see "Things to prepare in advance of cell isolation and cell culture").
  3. Add 10 μL of Anti-O4-APC.
  4. 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.
  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 prepared medium as a drop in the middle of each well of a coated 24-well plate (see "Things to prepare in advance for cell isolation 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. Maintain the culture by replacing 50% of prepared medium every other day.
    ▲Note: If oligodendrocytes are cultivated for longer periods, e.g., 2 weeks, we recommend increasing FGF-2 and PDGF-AA concentrations to 20 ng/mL instead of 10 ng/mL.

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 Anti-O4 pure antibody in staining buffer to the cells with a final concentration of 5–10 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 (anti-mouse IgM) 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.
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Cultured oligodendrocytes from neonatal mouse brain tissue. Neonatal whole mouse brains were dissociated using a Neural Tissue Dissociation Kit. Subsequently, oligodendrocytes were isolated using the Anti-O4 MicroBeads and O4-positive oligodendrocytes were cultured in MACS® Neuro Medium, MACS NeuroBrew®-21, 1% P/S, and 0.5 mM L-glutamine (+ PDGF-AA and FGF-2) on PLL-coated glass coverslips (5×104 cells per well of a 24-well plate). After 5 days, in vitro cells were fixed and stained with the oligodendrocyte-specific antibody Anti-MBP (green).

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