Application protocol
Isolation and cultivation of CD171 (L1CAM)-positive neurons from neonatal mouse brain
Materials
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.
| Column | Max. number of labeled cells | Max. number of total cells | Separator |
|---|---|---|---|
| Positive selection | |||
| MS | 1×10⁷ | 2×10⁷ | MiniMACS™, OctoMACS™, VarioMACS, SuperMACS II |
| LS | 2×10⁷ | 4×10⁷ | MidiMACS™, QuadroMACS, VarioMACS, SuperMACS II |
| Depletion | |||
| LD | 1.5×10⁷ | 3×10⁷ | MidiMACS, QuadroMACS, VarioMACS, SuperMACS II |
| Positive selection or depletion | |||
| autoMACS | 5×10⁷ | 1×108 | autoMACS Pro |
- 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
Protocol
Preparation of buffer for cell 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). 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).
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
Good to know
Isolate CD171 (L1CAM)-positive neurons from the single-cell suspension using the CD171 (L1CAM) MicroBead Kit. Follow the protocol of the kit data sheet.
Download data sheet
To stain and analyze the isolated neurons by flow cytometry, follow the protocol on cell surface flow cytometry staining.
- 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").
- Let the cells settle down for 30 minutes at 37 °C in the incubator.
- Carefully add 450 μL of prepared medium to each well.
- Take off the medium to remove non-attached and dead cells.
- Add 500 µl of prepared medium.
- 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
- Wash cells 3× with PBS.
- Fix cells with 2% PFA for 10 minutes at room temperature.
- 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. - Add staining buffer (see "Things to prepare in advance") and incubate for 10 minutes at room temperature.
- Discard staining buffer.
- 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.
- Wash cells 3× with autoMACS Running Buffer.
- 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.
- Wash cells 3× with autoMACS Running Buffer.
▲Note: For co-staining with additional antibodies repeat step 6–9. - Store cells in autoMACS Running Buffer.
- 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 Antibody | Secondary Antibody | |||||
|---|---|---|---|---|---|---|
| Antibody | Incubation temperature | Incubation time | Recommended antibody concentration | Secondary antibody | Incubation temperature | Incubation time |
| Anti-ACSA-2 | Room temperature | 10 minutes | 1–5 µg/mL | Anti-rat IgG2b | Room temperature | 10 minutes |
| Anti-GLAST (ACSA-1) | Room temperature | 10 minutes | 1–5 µg/mL | Anti-mouse IgG2a | Room temperature | 10 minutes |
| Anti-O4 | Room temperature | 10 minutes | 5–10 µg/mL | Anti-mouse IgM | Room temperature | 10 minutes |
| Anti-PSA-NCAM | Room temperature | 10 minutes | 5–10 µg/mL | Anti-mouse IgM | Room temperature | 10 minutes |
| CD11b | Room temperature | 10 minutes | 5–10 µg/mL | Anti-rat IgG2bκ | Room temperature | 10 minutes |
| CD68 | 2–8 °C | Overnight | 5–10 µg/mL | Anti-rat IgG2a | Room temperature | 1 hour |
| CD171 (L1CAM) | 2–8 °C | Overnight | 5–10 µg/mL | Anti-rat IgG2a | Room temperature | 1 hour |
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