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Neurogenesis occurs at different stages of mammalian development. Central nervous system (CNS) development begins with the induction of the neuroectoderm to form the neural plate and then the neural tube. The caudal region of the neural tube gives rise to the spinal cord and the rostral region becomes the brain. During these early stages of neural development, cells divide rapidly. Immature neurons migrate from zones of cell proliferation to their final positions and start to extend neurites to form contacts with other cells. After prenatal development, neurogenesis is limited and restricted to the subventricular zone in the lateral wall of the lateral ventricle, and the subgranular zone of the dentate gyrus of the adult mammalian brain. There, neural stem cells continuously generate new neurons throughout life.
Neural cells in the central nervous system
The CNS includes two main classes of cells: neurons and glial cells. Neurons receive, process, and transmit information, while glial cells support and protect neurons by maintaining homeostasis, forming myelin, supplying nutrients, destroying pathogens, and removing dead neurons, among other functions. In vertebrates, glial cells outnumber neurons 10–50 times, and are divided into microglia and macroglia. The two major classes of macroglia in the CNS are astrocytes and oligodendrocytes.
MACS Handbook:
Brain (mouse)
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Neural stem cells
Neurogenesis persists in two regions of the mammalian adult brain: the subventricular zone in the lateral wall of the lateral ventricle and in the subgranular zone of the dentate gyrus. Adult neural stem cells (NSCs) in these regions have the capacity to self-renew and generate new neurons throughout life. NSCs are a subset of astrocytes and thus express characteristic astrocyte markers such as GLAST and GFAP.
Neurons
The mammalian brain contains between 100 million and 100 billion neurons, depending on the species. Neurons are electrically excitable cells that transmit information through action potentials and neurotransmitters to other neurons or other cell types. Depending on the neurotransmitter and receptors on the receiving cell, the signal may be excitatory or inhibitory. Neurons exhibit extensive morphological and physiological heterogeneity and lack a specific pan cell surface marker, such that pan neuronal cells can only be identified and isolated indirectly by labeling non-neuronal cells.
Astrocytes
Astrocytes are the most abundant cell type of the CNS, and exhibit broad morphological and functional heterogeneity. Astrocytes regulate ion and glutamate homeostasis, control the number and function of synapses, contribute to wound healing, form the blood-brain–barrier, and modulate cerebral blood flow. Astroglial cells also act as adult neural stem cells in the subventricular zone of the lateral ventricle’s lateral wall and in the subgranular layer of the dentate gyrus. GLAST is the most abundant glutamate transporter and a specific astrocyte marker in the developing and neonatal mammalian CNS. Radial glia and stem cells also express GLAST. The ACSA-2 antigen is specifically expressed on GLAST (ACSA-1)-positive astrocytes and is a second astrocyte-specific surface marker (PMID: 28317180). The antigen targeted by Anti-ACSA-2 is ATP1B2 (PMID: 28373281).
Oligodendrocytes
Oligodendrocytes form the myelin sheath around neuronal axons, which facilitates the fast saltatory propagation of action potentials. Several distinct phenotypic developmental stages of oligodendrocytes have been identified, both in vivo and in vitro, based on the expression of specific markers. Morphological criteria alone are often insufficient to characterize each stage. Oligodendrocyte precursor cells (OPCs) are small, round, process-bearing cells with active proliferation and migratory properties. They are identified in vitro with the Anti-A2B5 antibody which recognizes several gangliosides. However, this antibody also labels other cell types in vivo, so more specific markers are used, such as the platelet-derived growth factor receptor alpha (PDGFRα – a transmembrane receptor tyrosine kinase), and the single membrane-spanning chondroitin sulphate proteoglycan NG2 (AN2). These are the most reliable markers for OPCs in vivo and are also used to label adult OPCs (PMID: 18931697). When OPCs begin to differentiate, they express a sulphated surface antigen known as POA (pro-oligodendroblast antigen), which is recognized by the Anti-O4 antibody. During oligodendrocyte development, O4 expression begins on late oligodendrocyte progenitors that are A2B5-positive. While A2B5 expression disappears, O4 expression continues. As the cells differentiate, they synthesize galactocerebroside, myelin basic protein (MBP), proteolipid protein (PLP), myelin oligodendrocyte glycoprotein (MOG), and O1.
Microglia
Microglia (brain macrophages) are the resident immune effector cells of the CNS. Activated microglia also serve as antigen-presenting cells. They are morphologically, immunophenotypically, and functionally related to cells of the monocyte/macrophage lineage and characterized by expression of CD11b in humans and mice (CD11b/c in rat), CD68, F4/80, and MHC class II.
Miltenyi Biotec has created dedicated applications to isolate, cultivate, and characterize different neural cell types.
A special protocol describing immunofluorescence staining with neural cell antibodies for microscopy, as well as tutorial videos for the isolation of viable astrocytes and microglia can be accessed in the Related resources panel to the right.
Neural cell samples can be obtained from whole brains, a brain region, or the spinal cord. Downstream applications to characterize neural cells may require prior sample preparation, such as tissue dissociation, to generate single-cell suspensions. For details, see Chapter mouse brain.
Miltenyi Biotec has developed numerous products for the isolation of distinct neural cell types from single-cell suspensions. Most kits are based on the positive selection of magnetically labeled target cells, but isolation of neurons is done by depleting non-target cells. For details on MACS® Cell Separation Technology, see the MACS Handbook chapter Magnetic cell separation .
MACS Handbook:
Magnetic cell separation
At a glance: Kits and reagents for the separation of neurons and neuronal precursor cells
| Starting material | Isolation strategy | Comments | Automation | Product |
|---|---|---|---|---|
| Neurons | ||||
| Single-cell suspension | Depletion of non-target cells | For neonatal or adult mouse brain dissociated with either the Neural Tissue Dissociation Kit – Postnatal neurons or the Adult Brain Dissociation Kit, mouse, rat | Yes* | Neuron Isolation Kit, mouse |
| Single-cell suspension | Positive selection of target cells | For neonatal mouse brain dissociated with the Neural Tissue Dissociation Kit (T) | Yes* | CD171 (L1CAM) MicroBead Kit, mouse |
| Neuronal precursor cells | ||||
| Single-cell suspension | Positive selection of target cells | For neonatal mouse or rat brain dissociated with the Neural Tissue Dissociation Kit (T) | Yes* | Anti-PSA-NCAM MicroBeads, human, mouse, rat |
| * Automated isolation can be performed on the autoMACS® Pro Separator | ||||
The ability to study neurons in a controlled environment and under specific conditions is necessary to understand cellular and molecular mechanisms during CNS development and in diseases. Existing techniques for isolation and cultivation of neurons have shortcomings. Cell culture with anti-mitotic agents (e.g. AraC) can lead to glial cell contamination, is time consuming and does not allow analysis of freshly isolated cells. Conventional flow cytometric, droplet-based cell sorting is relatively fast, but requires transgenic mice, and cells undergo pressure during the sorting process, which causes a reduced viability.
Miltenyi Biotec’s Neuron Isolation Kit, mouse is the only commercially available product for neuron isolation from both wild type and transgenic mice. Based on depletion of non-neuronal cells based on novel non-neuronal cell markers, the kit achieves purities of 97% for animal age ≤P7 and 93% for animal age >P7. The isolated neurons are ready for downstream applications, including cell culture, cellular and molecular analysis, and functional assays.
Adult neurons are especially fragile, making the isolation and culture of primary adult neurons challenging. Currently, isolation and culture are generally limited to embryonic tissues (approx. E16–18), when glial cells are less abundant, or at early postnatal stages before formation of synapses occurs. However, embryonic neurons behave differently from adult neurons in terms of pharmacology, electrophysiology, development, and regenerative and pathological characteristics. The Neuron Isolation Kit, mouse combined with the Adult Brain Dissociation Kit, mouse and rat and the gentleMACS™ Octo Dissociator with Heaters, allows isolation of highly purified viable neurons from adult mouse brain within half a day.
To assess and further boost purity of isolated neurons, Miltenyi Biotec offers a wide range of antibodies specific to non-neuronal cell surface markers to exclude contamination by glial cells, such as astrocytes, microglia, and oligodendrocytes.
Neuron isolation based on positive selection of CD171. P7 mouse cerebelli were dissociated using the Neural Tissue Dissociation Kit (T). Subsequently, neurons were isolated using the CD171 (L1CAM) MicroBead Kit, mosue. Cells were fluorescently stained with Labeling Check Reagent-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.
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At a glance: Kits and reagents for the separation of astrocytes
| Starting material | Isolation strategy | Comments | Automation | Product |
|---|---|---|---|---|
| Single-cell suspension | Positive selection of target cells | For dissociated neonatal or adult mouse brain | Yes | Anti-ACSA-2 MicroBead Kit, mouse |
| Single-cell suspension | Positive selection of target cells | For neonatal mouse or rat brain dissociated with the Neural Tissue Dissociation Kit (T) | Yes | Anti-GLAST (ACSA-1) MicroBead Kit, human, mouse, rat |
| PBS-based buffer optimized to remove dead cells during isolation of adult astrocytes | AstroMACS Separation Buffer |
Highly purified viable astrocytes from neonatal and adult mouse brain can be acquired by positive selection using one of the two astrocyte-specific cell surface markers, glutamate-aspartate transporter 1 (GLAST-1, also known as ACSA-1) or astrocyte cell surface antigen-2 (ACSA-2). Both antibodies (Anti-GLAST (ACSA-1) and Anti-ACSA-2) can be used for flow cytometry, immunocytochemistry, and immunohistochemistry analysis.
The Anti-GLAST (ACSA-1) antibody has been developed by Miltenyi Biotec and detects the extracellular epitope of the glutamate transporter GLAST-1 (EAAT1). This antibody was generated by immunization of GLAST-1 knockout mice with GLAST-expressing 1881 cells. The Anti-GLAST antibody labels all GFAP, GS, BLBP-positive astrocytes, reactive astrocytes, radial glia, Müller glia, and Bergmann glia, and shows superior performance in astrocyte detection and separation (PMID: 22374709).
The Anti-ACSA-2 antibody is a novel monoclonal antibody generated by immunizing rats with astrocytes isolated from GFAP-EGFP transgenic mice. Immunohistochemical and flow cytometry analyses demonstrate the high specificity of Anti-ACSA-2 for astrocyte lineage cells (PMID: 28317180). The ACSA-2 epitope was identified recently as ATP1B2 and the authors recommend the Anti-ACSA-2 antibody as “a first-choice method for astrocyte isolation and characterization.” (PMID: 28373281). Unlike GLAST, the ACSA-2 epitope is papain-resistant. Thus, it can be used for astrocyte separation and analysis after papain-based tissue dissociation, as well as for astrocyte isolation from adult mouse brain.
Related PDFs:
MACS Cell Separation - Select the best (brochure)
Anti-GLAST (ACSA-1) (brochure)
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At a glance: Kits and reagents for the separation of oligodendrocyte lineage cells
| Starting material | Isolation strategy | Comments | Automation | Product |
|---|---|---|---|---|
| Oligodendrocyte precursor cells (OPCs) | ||||
| Single-cell suspension | Positive selection of target cells | Use neonatal mouse brain | Yes | CD140a (PDGFRα) MicroBead Kit, mouse |
| Single-cell suspension | Positive selection of target cells | Use neonatal mouse brain; re-expression of antigen necessary | Yes | Anti-AN2 (NG2) MicroBeads, human, mouse |
| Single-cell suspension | Positive selection of target cells | Use neonatal mouse brain | Yes | Anti-A2B5 MicroBeads, human, mouse, rat |
| Premature oligodendrocytes | ||||
| Single-cell suspension | Positive selection of target cells | Use neonatal or adult mouse or rat brain | Yes | Anti-O4 MicroBeads, human, mouse, rat |
Miltenyi Biotec offers a range of MicroBeads and kits for the isolation of oligodendrocyte lineage cells. Anti-A2B5 MicroBeads, human, mouse, rat enable selection of glial progenitor cells, whereas the CD140a (PDGFRα) MicroBead Kit and Anti-AN2 (NG2) MicroBeads serve to isolate oligodendrocyte precursors.
Premature oligodendrocytes can be isolated from neonatal and adult mouse brain tissue with the Anti-O4 MicroBeads, human, mouse, rat.
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At a glance: Kits and reagents for the separation of microglia
| Starting material | Isolation strategy | Comments | Automation | Product |
|---|---|---|---|---|
| Single-cell suspension | Positive selection of target cells | Use neonatal or adult mouse brain | Yes | CD11b (Microglia) MicroBeads, human and mouse |
| Single-cell suspension | Positive selection of target cells | Use neonatal or adult rat brain | Yes | CD11b/c (Microglia) MicroBeads, rat |
Highly purified (99%) and viable microglial cells can be separated from both neonatal and adult mouse or rat brain tissue with CD11b (Microglia) MicroBeads, human and mouse or CD11b/c (Microglia) MicroBeads, rat. In combination with the MultiMACS™ Cell24 Separator Plus or autoMACS Pro Separator, these kits enable fast microglial cell isolation from up to 24 samples in parallel.
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Related videos:
At a glance: Kits and reagents for the separation of endothelial cells
| Starting material | Isolation strategy | Comments | Automation | Product |
|---|---|---|---|---|
| Single-cell suspension | Depletion of non-target cells followed by positive selection of target cells | Use neonatal or adult mouse brain, and the Neural Tissue Dissociation Kit (P) | Yes | CD31 MicroBeads, mouse and CD45 MicroBeads, mouse |
Highly purified endothelial cells can be acquired from both neonatal or adult mouse brain by first depleting non-target cells with CD45 MicroBeads, mouse, and then selecting endothelial cells using the CD31 MicroBeads, mouse.
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At a glance: Antibodies for flow cytometry analysis of human, mouse, or rat neural cells
| Cell type | Antibody | Fluorochrome |
|---|---|---|
| Astrocytes | • Anti-ACSA-2, mouse (clone: IH3-18A3) • Anti-ACSA-2, mouse (clone: REA969) • Anti-GLAST (ACSA-1), human, mouse, rat (clone: ACSA-1) • Anti-GFAP, human, mouse, rat (clone: REA335) | Multiple |
| Oligodendrocytes and OPCs | • Anti-O4, human, mouse, rat (clone: REA576) • Anti-O4, human, mouse, rat (clone: O4) • Anti- AN2(NG2), human, mouse (clone: 1E6.4) • Anti-CD140a (PDGFRa), mouse (clone: APA5) • Anti-CD140a (PDGFRa), mouse (clone: REA637) • Anti-A2B5, human, mouse, rat(clone: 105HB29) | Multiple |
| Microglia | • Anti-CD11b, human, mouse (clone: REA592) • Anti-CD11b, human and mouse (clone: M1/70.15.11.5) • Anti-CD11b/c, rat (clone: REA325) • Anti-CD68, mouse (clone: FA-11) • Anti-CD68, rat (clone: REA237) • Anti-F4/80, mouse (clone: REA126) | Multiple |
| Neurons and neuronal precursor cells | • CD171 (L1CAM), mouse (clone: 555) • CD171 (L1CAM), human (clone: REA163) • CD271 (NGFR) , mouse and human (clone: REA648) • Anti-PSA-NCAM, human, mouse, rat (clone: 2-2B) • Anti-PAX-6, human (clone: REA507) | Multiple |
| Endothelial cells | • CD31, mouse (clone: 390) | Multiple |
Miltenyi Biotec offers a broad range of validated antibodies against cell-specific surface markers for flow cytometry. These antibodies enable the determination of the purity of isolated cells, quantification of cell populations, and examination of overlapping markers. Analysis of the oligodendrocyte cell lineage is a good example of the discriminatory power of this characterization method. A2B5 expression decreases with animal age as oligodendrocytes mature. Flow cytometry analysis using Anti-AN2 and Anti-A2B5 monoclonal antibodies can show changes in expression and enable quantification of oligodendrocyte and glial precursor cells.
At a glance: Kits and reagents for the cultivation of neural cells
| Use | Comments | Product |
|---|---|---|
| Basal medium | MACS Neuro Medium | |
| Supplement | Serum-free and optimized for long-term mature neuron culture. Can also be used for oligodendrocyte culture. | MACS NeuroBrew-21 |
| Supplement | Vitamin A induces differentiation of neural stem cells. Therefore, MACS NeuroBrew-21 w/o Vitamin A is designed for use with primary neural stem or progenitor cells, primary neurospheres, ES/iPS cell–derived neural stem cells and neurospheres to maintain cells in an undifferentiated state. | MACS NeuroBrew-21 w/o Vitamin A |
| Astrocyte culture medium | Serum-free; includes MACS Neuro Medium, MACS NeuroBrew-21, and AstroMACS Supplement, which is optimized for astrocyte survival and growth. | AstroMACS Medium |
Cultivation of primary neurons and glial cells is widely used to study brain function in a controlled in vitro environment. This is a powerful tool to understand the molecular and cellular mechanisms of neural cell dysfunction and death in neurodegenerative diseases; to study the individual contribution of different cell types to disease progression; and to investigate the interactions between neural cells during development and disease.
MACS Neuro Medium supplemented with the serum-free MACS NeuroBrew®-21 are optimized to support growth and long-term survival of mouse neonatal and adult neurons, astrocytes, and oligodendrocytes. To maintain undifferentiated neural stem or precursor cells, MACS NeuroBrew-21 is also available without vitamin A (MACS NeuroBrew-21 w/o Vitamin A).
Miltenyi Biotec's serum-free AstroMACS Medium supports neonatal astrocyte growth even at very low seeding densities, AstroMACS Medium also enhances cultivation of adult astrocytes, especially when AstroMACS Separation Buffer is used during prior cell separation to remove dead cells and provide a healthy culture environment.
For more information about Miltenyi Biotec media optimized for neural cells, see chapter Cell culture.
Miltenyi Biotec offers an extensive portfolio of cytokines for neural cell differentiation and maintenance, including human BDNF, CTNF, EGF, FGF-2, and GDNF, which can be purchased in premium, research or MACS GMP Grade.
MACS Handbook:
Cell culture
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At a glance: Antibodies for immunocytochemistry analysis of different neural cell types
| Cell type | Commonly used markers | Available from Miltenyi Biotec | ||
|---|---|---|---|---|
| Antibody | Clone | Isotype | ||
| Astrocytes | GLAST (EAAT1), ACSA-2, GFAP, ALDH1L1, S100β | • Anti-ACSA-2 pure, mouse • Anti-GLAST (ACSA-1) pure, human, mouse, rat | • IH318A3 • ACSA-1 | • Rat IgG2b • Mouse IgG2a |
| Mature/premature oligodendrocytes | O4, AN2(NG2), PDGFRα(CD140a), A2B5, MOG, MAG, PLP, MBP, O1 | • Anti-O4 pure, human, mouse, rat | • O4 | • Mouse IgM |
| Microglia | CD11b or CD11b/c, CD68, F4/80, CD45, IBA1 | • CD11b pure, human and mouse • CD68 pure, mouse | •M1/70.15.11.5 • FA11 | • Rat IgG2bκ • Rat IgG2a |
| Neurons | MAP2, TuJ1, NeuN, β-III Tubulin, NFH, CD171 ( L1CAM) | |||
| Neuronal stem/ precursor cells | PSA-NCAM | • Anti-PSA-NCAM pure, human, mouse, rat | • 2-2B | • Mouse IgM |
| Subtype of neuronal cells (e.g. Purkinje cells) | • CD171 (L1CAM) pure, mouse | • 555 | • Rat IgG2a | |
Immunohistochemistry (IHC) or immunocytochemistry (ICC) techniques have contributed immensely to the advancement of neuroscience. By staining cells with highly specific, fluorescent probes, scientists can visualize brain tissue structure, cellular organization, and molecular and cellular architecture in high-resolution, color images.
A key element for successful and meaningful IHC or ICC staining are antibodies that bind with great specificity to the antigen of interest. This specificity enables the usage of probes to distinguish between neurons, astrocytes, oligodendrocytes, and other cells, and to assess the purity of isolated cell populations and cell cultures. Both polyclonal or monoclonal antibodies can be used for ICC or IHC. However, monoclonal antibodies are preferred, due to their higher specificity, purity, consistency, and lower background.
Miltenyi Biotec has developed monoclonal antibodies for specific immunofluorescent staining of cultivated adherent neural cells or PFA-fixed tissue cryosections.
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