Application protocol

Isolation and cultivation of CD171 (L1CAM)-positive neurons from neonatal mouse brain

The CD171 (L1CAM) (L1 cell adhesion molecule) antigen is expressed by tetanus-toxin positive neurons, endothelial cells, certain epithelial cells, reticular fibroblasts, and several malignant tumors, including colon and breast carcinomas, colon melanoma, and tumor cells of neuronal and mesothelial origin. CD171 plays a vital role in cell adhesion and signal transduction, and has been demonstrated to play a role in augmenting tumor growth. It is involved in the development of the nervous system and regulates processes such as neuron–neuron adhesion, myelination, axonal guidance, and neuronal migration. CD171 (L1CAM) MicroBeads are designed for the separation of neurons based on the expression of CD171 (L1CAM), and especially optimized for use with dissociated postnatal CD-1® mouse brain tissue derived from animals younger than postnatal day eight (P8). Up to 99.5% purity can be achieved with P7 cerebellum tissue because the expression of CD171 is found almost exclusively on neurons. Purity decreases with age: P9 up to 80%; P12 up to 64%. For other brain regions, glial cells may need to be depleted if high purities are required. The CD171 (L1CAM) epitope shows papain sensitivity. Therefore, the Neural Tissue Dissociation Kit (T) is recommended to generate a single-cell suspension.

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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.

Reagent and instrument requirements

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

▲ Note: Column adapters are required to insert certain columns into the VarioMACS™ or SuperMACS™ II Separators. For details refer to the respective MACS Separator data sheet.

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

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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.

1. Things to prepare in advance of cell isolation and cell culture

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.

2. Dissociation of brain tissue

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.

3. Isolation of CD171 (L1CAM)-positive neurons

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

CD171 (L1CAM) MicroBead Kit, mouse

4. Flow cytometry analysis

To stain and analyze the isolated neurons by flow cytometry, follow the protocol on cell surface flow cytometry staining.

5. Cell culture

Plate 5×10⁴cells 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.

6. Immunocytochemical staining of cultured neurons

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

View details

Successful culture of CD171 (L1CAM)-positive neurons isolated from neonatal mouse brain tissue. P7 mouse cerebelli were dissociated using the Neural Tissue Dissociation Kit, and CD171-positive cells were isolated from the single-cell suspension using the CD171 MicroBead Kit. After 5 days culture in MACS® Neuro Medium, MACS NeuroBrew®-21, 1% P/S, and 0.5 mM L-glutamine, cells were fixed and stained with the neuron-specific antibodies Anti-MAP2 (red) and Anti-TuJ1 (green).

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