Name: isolation of distinct cell populations from the developing cerebellum by microdissection
Uploaded: 2014-09-21T15:00:00.000+00:00
Duration: 8 min 2 s
Description: Watch this Scientific Journal Video about Isolation of Distinct Cell Populations from the Developing Cerebellum by Microdissection at JoVE.com

The authors would like to thank James Oesterling for flow cytometry assistance. This research was supported by a grant from the US National Cancer Institute (R01-CA178380, Z.Y.) and a US National Institutes Postdoctoral training grant (5T32CA009035-37, L.W.Y.).

Use of animals in this protocol has been performed in accordance with procedures approved by the Fox Chase Cancer Center Animal Care and Use Committee.
1. Preparation of Instruments, Solutions, and Coverslips
<ol>
	<li>Clean two sets of #5 fine forceps, one set of #7 fine curved forceps, one large surgical scissor, one microdissecting scissor, one spatula and one perforated spoon. Place the instruments in a self-sealing sterilization pouch and autoclave. Keep instruments in pouch until ready for use.</li>
	<li>Prepare a 3% low melting temperature agarose solution.
	<ol>
		<li>Add 1.5 g agarose into 50 ml of sterile Dulbecco&#8242;s Phosphate Buffered Saline (DPBS) then place in microwave and heat until bubbles appear. Swirl flask and reheat until all agarose has dissolved. Mix solution with a stir bar prior to heating if clumps appear.</li>
		<li>Store solution in a 37 &#176;C water bath until needed.</li>
		<li>For long-term storage, store the agarose solution for future use at room temperature or at 4 &#176;C for months. Reheat in microwave to liquefy. For repeated heating of the agarose solution, weigh the flask before heating to maintain its initial weight with warm sterile distilled water.</li>
	</ol>
	</li>
	<li>Prepare the following solutions for papain-based cell dissociation as previously described2:
	<ol>
		<li>A papain solution with papain (100 U/ml), cysteine (0.2 mg/ml) and DNase (250 U/ml) in sterile phenol red-containing DPBS.</li>
		<li>An ovomucoid solution with bovine serum albumin (BSA; 8 mg/ml), soybean trypsin inhibitor (8 mg/ml)) and DNase (250 U/ml) in sterile phenol red-containing DPBS. 
		Filter sterilize both solutions and adjust pH to return original color of phenol red-containing DPBS.</li>
	</ol>
	</li>
	<li>Prepare cell culture media (NB/B27): supplement basal media with 1 mM sodium pyruvate, 2 mM L-glutamine, 1% penicillin-streptomycin (P/S) and B27 supplement. Filter sterilize and protect from light. Equilibrate media at 37 &#176;C and 5% CO2 before use.</li>
	<li>Prepare FACS buffer: 5% fetal bovine serum (FBS) in DPBS. Add 2.5 ml FBS to 47.5 ml DPBS.</li>
	<li>Prepare poly-D-lysine (PDL)-coated coverslips.
	<ol>
		<li>Autoclave new glass coverslips.</li>
		<li>Coat the coverslips with PDL (100 &#956;g/ml in sterile distilled water) and incubate for 2 hr at 37 &#176;C or room temperature overnight.</li>
		<li>Wash coverslips with sterile distilled water. Wash once with&#160;NB/B27 media. Let coverslips dry completely in tissue culture hood prior to use.</li>
	</ol>
	</li>
</ol>
2. Dissection and Preparation of Tissue Slices
<ol>
	<li>On the day of dissection, wipe down the dissection area with 70% ethanol and cover with an absorbent pad.</li>
	<li>Remove dissection tools from the sterilization pouch. Add ice cold, sterile DPBS/1% P/S to a small Petri dish and place on ice.</li>
	<li>Decapitate P4 Math1-GFP/Nestin-CFP mouse pups with large surgical scissors then hold the head down with fine curved forceps. Remove the skin and gently peel away the skull with #5 fine forceps then scoop out the brain from the skull using a spatula and transfer it to the Petri dish containing DPBS/1% P/S.
	<ol>
		<li>Using #5 fine forceps, carefully separate the cerebellum from the rest of the brain. Be sure to dissect one pup at a time as quickly as possible.</li>
		<li>Collect a minimum of 8 cerebella in order to obtain enough NEPs for molecular and functional analyses.</li>
	</ol>
	</li>
	<li>Fill an embedding mold measuring 2 x 2 x 2 cm with 3% low melting temperature agarose solution. Ensure that the temperature of agarose is no more than 37 &#176;C.</li>
	<li>Remove excess liquid from cerebellum by dabbing gently on a laboratory tissue. Place cerebellum into mold in a vertical position. Place the mold on ice to allow it to solidify quickly. Use ~4 cerebella per block.</li>
	<li>Obtain living tissue sections with a vibrating blade vibratome. Trim cerebella-containing agarose block with a razor blade. Glue the agarose block to the vibratome plate with cerebella oriented in a vertical position.</li>
	<li>Fill the tray of the vibratome with ice-cold sterile DPBS/1% P/S and place ice underneath.
	<ol>
		<li>Cut 600 &#956;m sections through the whole length of cerebellum. Collect sections from the ice tray using a perforated spoon. Place sections in a Petri dish filled with DPBS/1% P/S on ice.</li>
	</ol>
	</li>
</ol>
3. Microdissection and Cell Dissociation
<ol>
	<li>Place the Petri dish containing tissue slices under a fluorescent dissecting microscope.
	<ol>
		<li>Carefully separate the EGL from the rest of the cerebellar section using fine forceps by dissecting in between the CFP+ molecular layer and the GFP+ EGL (refer to dotted line in Figure 1). Be careful to not include any part of the molecular layer, which will result in contamination of Nestin-expressing Bergmann glia.</li>
		<li>Complete this step as quickly as possible and keep the tissue on ice to avoid cell death.</li>
		<li>Remove the agarose surrounding the tissue before proceeding to the next step.</li>
	</ol>
	</li>
	<li>Digest the microdissected EGL tissue using a papain-based protocol as previously described2 to obtain a single cell suspension.
	<ol>
		<li>Re-suspend cells in FACS buffer for collection of CFP-positive cells via FACS.</li>
	</ol>
	</li>
</ol>
4. Cell Sorting and Plating
<ol>
	<li>Sort cells for CFP fluorescence using a sterile, high speed flow cytometer containing an appropriate CFP filter and collect cells in FACS buffer on ice. Obtain approximately 100,000 NEPs from each P4 cerebellum.</li>
	<li>Centrifuge cells at 300 x g for 5 min. Use cells immediately for molecular analysis or culture for further experimentation.</li>
	<li>To culture cells, re-suspend the cells in pre-warmed NB/B27 media and plate on PDL-coated coverslips as previously described2.</li>
</ol>

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P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+neuronal+precursor+proliferation+in+the+cerebellum+by+Sonic+Hedgehog.">Control of neuronal precursor proliferation in the cerebellum by Sonic Hedgehog.</a> Neuron. 22, 103-114 (1999).</li><li>Dahmane, N., Ruizi Altaba, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sonic+hedgehog+regulates+the+growth+and+patterning+of+the+cerebellum.">Sonic hedgehog regulates the growth and patterning of the cerebellum.</a> Development. 126, 3089-3100 (1999).</li><li>Riobo, N. A., Manning, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathways+of+signal+transduction+employed+by+vertebrate+Hedgehogs.">Pathways of signal transduction employed by vertebrate Hedgehogs.</a> Biochem J. 403, 369-379 (2007).</li><li>Dahmane, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Sonic+Hedgehog-Gli+pathway+regulates+dorsal+brain+growth+and+tumorigenesis.">The Sonic Hedgehog-Gli pathway regulates dorsal brain growth and tumorigenesis.</a> Development. 128, 5201-5212 (2001).</li><li>Marino, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Medulloblastoma:+developmental+mechanisms+out+of+control.">Medulloblastoma: developmental mechanisms out of control.</a> Trends Mol Med. 11, 17-22 (2005).</li><li>Yang, Z. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Medulloblastoma+can+be+initiated+by+deletion+of+Patched+in+lineage-restricted+progenitors+or+stem+cells.">Medulloblastoma can be initiated by deletion of Patched in lineage-restricted progenitors or stem cells.</a> Cancer Cell. 14, 135-145 (2008).</li><li>Goodrich, L. V., Milenkovic, L., Higgins, K. M., Scott, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Altered+neural+cell+fates+and+medulloblastoma+in+mouse+patched+mutants.">Altered neural cell fates and medulloblastoma in mouse patched mutants.</a> Science. 277, 1109-1113 (1997).</li><li>Li, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+population+of+Nestin-expressing+progenitors+in+the+cerebellum+exhibits+increased+tumorigenicity.">A population of Nestin-expressing progenitors in the cerebellum exhibits increased tumorigenicity.</a> Nat Neurosci. 16, 1737-1744 (2013).</li><li>Ben-Arie, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Math1+is+essential+for+genesis+of+cerebellar+granule+neurons.">Math1 is essential for genesis of cerebellar granule neurons.</a> Nature. 390, 169-172 (1997).</li><li>Sutter, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cerebellar+stem+cells+act+as+medulloblastoma-initiating+cells+in+a+mouse+model+and+a+neural+stem+cell+signature+characterizes+a+subset+of+human+medulloblastomas.">Cerebellar stem cells act as medulloblastoma-initiating cells in a mouse model and a neural stem cell signature characterizes a subset of human medulloblastomas.</a> Oncogene. 29, 1845-1856 (2010).</li><li>Sotelo, C., Alvarado-Mallart, R. M., Frain, M., Vernet, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+plasticity+of+adult+Bergmann+fibers+is+associated+with+radial+migration+of+grafted+Purkinje+cells.">Molecular plasticity of adult Bergmann fibers is associated with radial migration of grafted Purkinje cells.</a> J Neurosci. 14, 124-133 (1994).</li><li>Lumpkin, E. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Math1-driven+GFP+expression+in+the+developing+nervous+system+of+transgenic+mice.">Math1-driven GFP expression in the developing nervous system of transgenic mice.</a> Gene Expr Patterns. 3, 389-395 (2003).</li><li>Encinas, J. 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The authors have nothing to disclose.

The microdissection method described here is the first procedure able to isolate a pure population of living cells for use in molecular and functional analyses. As demonstrated by Li et al.9, NEPs obtained by this method can be cultured and incorporated into various experiments and because of their high purity, can also be used for microarray analysis.
It is very important to take time to become proficient with this microdissection technique. NEPs are located in the deep region of the EGL in very close proximity to the molecular layer, in which Nestin-expressing Bergmann glia could contaminate the NEP cell isolation and compromise purity. Microdissection should therefore be done conservatively to make sure contaminating cells in adjacent tissue are not included. The major advantage of the microdissection technique described here is the ability to obtain living cells that can be cultured for further experimentation. Because of this, it is crucial that all steps during this procedure be done quickly and that tissue be kept on ice as much as possible. Also, all instruments and reagents must be sterile. Following these steps will reduce cell death and cell culture contamination. In addition, mounting multiple cerebella per agarose block will aid in reducing the procedure time.
This technique is not limited to isolation of NEPs and can be used to collect cells from other areas of the cerebellum, such as glial cells from the molecular layer and neural stem cells from the white matter. Beside the cerebellum, microdissection can be used with other tissues that contain recognizable regions, such as the hippocampus, for example. The use of fluorescent transgenes, as utilized here, can aid in creating borders or identify regions for dissection, which can be especially helpful in tissues that lack easily identifiable areas and would otherwise not be able to be microdissected. This technique could therefore aid in cell purifications in a wide range of research areas and applications.

The cerebellum is comprised of multiple cell layers, each containing distinct cell types. During development, the EGL contains proliferating granule neuron precursors (GNPs) while the molecular layer and Purkinje layer contain Bergmann glia and Purkinje neurons, respectively. Deep within the cerebellum lies the white matter, which contains neural stem cells (NSCs) and oligodendrocytes1.
Sonic hedgehog (Shh) plays a critical role in regulating cerebellar development and in particular, it promotes the proliferation of GNPs via binding to its receptor Patched (Ptc), a negative regulator of Shh signaling2-4. Aberrant activation of Shh signaling generates medulloblastoma (MB), the most common malignant brain tumor in children5,6.
Ptc mutant MB transgenic mouse models are powerful tools for the study of MB and develop tumors resembling human MB7,8. Using these mice, it was discovered that GNPs are the cell of origin for hedgehog-type MB7. Furthermore, in addition to GNPs, we have recently identified a unique population of neuronal progenitors within the EGL of the developing cerebellum that can also give rise to MB. These cells express high levels of the type VI intermediate filament protein, Nestin, and are termed NEPs9. NEPs are located within the deep part of the EGL and exist transiently only during neonatal cerebellar development. They are committed to the granule cell lineage as GNPs but are distinct as they are quiescent and do not express Math1, a well-established marker for conventional GNPs10. In addition, NEPs give rise to MB more efficiently than GNPs after activation of the Shh signaling pathway9, which makes them a novel origin for MB tumorigenesis.
We previously isolated NEPs from the cerebellar EGL at postnatal day 4 (P4) by the microdissection technique described here9. Microdissection via direct microscopic visualization of the tissue allows for specific dissection of the cerebellar EGL. This is necessary as Nestin is also expressed by NSCs in the cerebellar white matter and by Bergmann glia in the molecular layer1,7,11,12 and it was crucial that these cells not be included, as they would confound analysis. Cells isolated from microdissected tissue can be used immediately for molecular analysis or they can be cultured for further applications.
To aid in specifically microdissecting the EGL and to further purify NEPs, Math1-GFP (green fluorescent protein) mice were crossed with Nestin-CFP (cyan fluorescent protein) mice. The transcription factor Math1 is specifically expressed by GNPs and GFP expression is clearly visible in the EGL9,13. Nestin-CFP mice express a nuclear form of CFP and can be easily visualized in the molecular layer9,14. Together, GFP and CFP expression create boundaries for microdissection of the EGL (see Figure 1). CFP-positive NEPs were then isolated by FACS, following enzymatic dissociation of the dissected EGLs.
Currently, the most well utilized method to isolate cells from the EGL is Percoll gradient centrifugation of whole cerebellar tissue15,16. This method, however, is unable to completely exclude Nestin+ cell populations from the molecular layer and white matter and therefore cannot be used for the study of NEPs. Laser capture microdissection, which uses the transfer of laser energy to remove cells of interest, is another method used to isolate specific cells within a heterogeneous tissue17,18 but captured cells can only be used for recovery of DNA, RNA and protein and are not able to be cultured.
This protocol provides a way to specifically isolate an alive, pure population of NEPs from the EGL. This technique can also be applied to different tissue types that have either recognizable anatomical architecture or fluorescently labeled cells/regions. Therefore, the major advantage of incorporating this novel microdissection technique into cell isolation protocols is that cells can be isolated from specific tissue regions to eliminate contaminating neighboring tissue and the cells can be collected immediately for analysis or cultured for other applications.

<table><tbody><tr><td>Neurobasal media</td><td>Gibco</td><td>21103-049</td><td></td></tr><tr><td>PDL</td><td>Millipore</td><td>A-003-E</td><td></td></tr><tr><td>Ultra pure low melting temperature agarose</td><td>Invitrogen</td><td>16520-050</td><td></td></tr><tr><td>DPBS</td><td>Gibco</td><td>14190144</td><td></td></tr></tbody></table>

isolation of distinct cell populations from the developing cerebellum by microdissection

Microdissection is a novel technique that can isolate specific regions of a tissue and eliminate contamination from cellular sources in adjacent areas. This method was first utilized in the study of Nestin-expressing progenitors (NEPs), a newly identified population of cells in the cerebellar external germinal layer (EGL). Using microdissection in combination with fluorescent-activated cell sorting (FACS), a pure population of NEPs was collected separately from conventional granule neuron precursors in the EGL and from other contaminating Nestin-expressing cells in the cerebellum. Without microdissection, functional analyses of NEPs would not have been possible with the current methods available, such as Percoll gradient centrifugation and laser capture microdissection. This technique can also be applied for use with various tissues that contain either recognizable regions or fluorescently-labeled cells. Most importantly, a major advantage of this microdissection technique is that isolated cells are living and can be cultured for further experimentation, which is currently not possible with other described methods.

As shown in Figure 1A, cerebellar slices were prepared from P4 Math1-GFP/Nestin-CFP mice, in which the cerebellar EGL was dominated by GFP-expressing GNPs and the molecular layer was enriched by CFP-positive glial cells. To isolate NEPs that are located in the deep part of the EGL, slices were microdissected between the EGL and the molecular layer (along dashed line; Figure 1A). Dissected EGLs (Figure 1B) were then collected for enzymatic dissociation followed by FACS.
As expected, a majority (more than 85%) of cells in the dissected EGL were conventional GNPs, which were positive for GFP (Figure 2A). NEPs (CFP+) account for only around 5% of the EGL cell population. Almost none of the cells were double-positive for GFP and CFP based on the FACS analysis. CFP+ cells purified by FACS, were cultured in vitro to examine the differentiation potential. As shown in Figure 2B, NEPs exclusively gave rise to &#946;-tubulin+ neurons after 4 days in culture, suggesting NEPs are lineage-restricted neuronal progenitors.
By this microdissection protocol, NEPs and GNPs were also purified from the EGL of Ptc deficient cerebellum. Upon intracranial transplantation, NEPs develop MBs more rapidly compared with GNPs, indicating that NEPs are particularly susceptible to oncogenic transformation9.
<img alt="Figure 1" fo:content-width="5in" src="/files/ftp_upload/52034/52034fig1highres.jpg" width="500" /> 
Figure 1. Identification of the region for EGL microdissection. Fluorescent images from P4 Math1-GFP/Nestin-CFP animals. (A) GFP expression is located in the EGL, while the majority of CFP expression is located in the molecular layer. Microdissection was done along the yellow dotted line. (B) EGLs were collected for tissue dissociation. This figure has been modified from Li et al9.
<img alt="Figure 2" fo:content-width="5in" src="/files/ftp_upload/52034/52034fig2highres.jpg" width="500" /> 
Figure 2. NEPs represent a small population of cells in the EGL and are lineage-restricted neuronal progenitors. (A) Flow cytometry analysis of CFP+ cells isolated from microdissected EGL. (B) NEPs were cultured under differentiation conditions for 4 days and stained for &#946;-tubulin (red) and counterstained with DAPI (blue). This figure has been modified from Li et al.9

Watch this Scientific Journal Video about Isolation of Distinct Cell Populations from the Developing Cerebellum by Microdissection at JoVE.com

Isolation of Distinct Cell Populations from the Developing Cerebellum by Microdissection

Nestin-expressing&#160;progenitors are a newly identified population of neuronal progenitors in the developing cerebellum. Using the microdissection technique presented here in combination with fluorescent-activated cell sorting, this cell population can be purified with no contamination from other cerebellar regions and can be cultured for further studies.

Microdissection is a novel technique that can isolate specific regions of a tissue and eliminate contamination from cellular sources in adjacent areas. ...

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Neuroscience

9.5K Views.  Temple University Health System. Nestin-expressing progenitors are a newly identified population of neuronal progenitors in the developing cerebellum. Using the microdissection technique presented here in combination with fluorescent-activated cell sorting, this cell population can be purified with no contamination from other cerebellar regions and can be cultured for further studies.

Video: Isolation of Distinct Cell Populations from the Developing Cerebellum by Microdissection

Ex Vivo Culture of Chick Cerebellar Slices and Spatially Targeted Electroporation of Granule Cell Precursors

The cerebellar external granule layer (EGL) is the site of the largest transit amplification in the developing brain, and an excellent model for studying neuronal proliferation and differentiation. In addition, evolutionary modifications of its proliferative capability have been responsible for the dramatic expansion of cerebellar size in the amniotes, making the cerebellum an excellent model for evo-devo studies of the vertebrate brain. The constituent cells of the EGL, cerebellar granule progenitors, also represent a significant cell of origin for medulloblastoma, the most prevalent paediatric neuronal tumour. Following transit amplification, granule precursors migrate radially into the internal granular layer of the cerebellum where they represent the largest neuronal population in the mature mammalian brain. In chick, the peak of EGL proliferation occurs towards the end of the second week of gestation. In order to target genetic modification to this layer at the peak of proliferation, we have developed a method for genetic manipulation through ex vivo electroporation of cerebellum slices from embryonic Day 14 chick embryos. This method recapitulates several important aspects of in vivo granule neuron development and will be useful in generating a thorough understanding of cerebellar granule cell proliferation and differentiation, and thus of cerebellum development, evolution and disease.

Ex Vivo Culture of Chick Cerebellar Slices and Spatially Targeted Electroporation of Granule Cell Precursors

The cerebellar external granule layer is the site of the largest transit amplification in the developing brain. Here, we present a protocol to target genetic modification to this layer at the peak of proliferation using ex vivo electroporation and culture of cerebellar slices from embryonic Day 14 chick embryos.

The cerebellar external granule layer (EGL) is the site of the largest transit amplification in the developing brain, and an excellent model for studying ...

Developmental Biology

Isolation and Culture of Post-Natal Mouse Cerebellar Granule Neuron Progenitor Cells and Neurons 

The cerebellar cortex is a well described structure that provides unique opportunities for studying neuronal properties and development1,2. Of the cerebellar neuronal types (granule cells, Purkinje cells and inhibitory interneurons), granule neurons are by far the most numerous and are the most abundant type of neurons in the mammalian brain. In rodents, cerebellar granule neurons are generated during the first two post-natal weeks from progenitor cells in the outermost layer of the cerebellar cortex, the external granule layer (EGL). The protocol presented here describes techniques to enrich and culture granule neurons and their progenitor cells from post-natal mouse cerebellum. We will describe procedures to obtain cultures of increasing purity3,4, which can be used to study the differentiation of proliferating progenitor cells into granule neurons5,6. Once the progenitor cells differentiate, the cultures also provide a homogenous population of granule neurons for experimental manipulation and characterization of phenomena such as synaptogenesis, glutamate receptor function7, interaction with other purified cerebellar cells8,9 or cell death7.

Isolation and Culture of Post-Natal Mouse Cerebellar Granule Neuron Progenitor Cells and Neurons

Here we present a method to isolate and culture cerebellar granule neuron progenitor cells and cerebellar granule neurons from postnatal mouse.

The cerebellar cortex is a well described structure that provides unique opportunities for studying neuronal properties and development1,2. Of the ...

Biology

Isolation of Cortical Microglia with Preserved Immunophenotype and Functionality From Murine Neonates

Isolation of microglia from CNS tissue is a powerful investigative tool used to study microglial biology ex vivo. The present method details a procedure for isolation of microglia from neonatal murine cortices by mechanical agitation with a rotary shaker. This microglia isolation method yields highly pure cortical microglia that exhibit morphological and functional characteristics indicative of quiescent microglia in normal, nonpathological conditions in vivo. This procedure also preserves the microglial immunophenotype and biochemical functionality as demonstrated by the induction of morphological changes, nuclear translocation of the p65 subunit of NF-&#954;B (p65), and secretion of the hallmark proinflammatory cytokine, tumor necrosis factor-&#945; (TNF-&#945;), upon lipopolysaccharide (LPS) and Pam3CSK4 (Pam) challenges. Therefore, the present isolation procedure preserves the immunophenotype of both quiescent and activated microglia, providing an experimental method of investigating microglia biology in ex vivo conditions.

One key to successful investigation of microglial biology is the preservation of microglial immunofunction ex vivo during isolation from CNS tissue. Isolating microglia via rotary shaking results in highly pure and immunofunctional cell cultures as assessed by fluorescent imaging, immunocytochemistry, and ELISA following microglia activation with the proinflammatory stimuli lipopolysaccharide (LPS) and Pam3CSK4 (Pam).

Isolation of microglia from CNS tissue is a powerful investigative tool used to study microglial biology ex vivo. The present method details a procedure ...

Immunology and Infection

Preparation and Immunostaining of Myelinating Organotypic Cerebellar Slice Cultures

In the nervous system, myelin is a complex membrane structure generated by myelinating glial cells, which ensheathes axons and facilitates fast electrical conduction. Myelin alteration has been shown to occur in various neurological diseases, where it is associated with functional deficits. Here, we provide a detailed description of an ex vivo model consisting of mouse organotypic cerebellar slices, which can be maintained in culture for several weeks and further be labeled to visualize myelin.

Here we present a method to prepare organotypic slice cultures from mouse cerebellum and myelin sheath staining by immunohistochemistry suitable for investigating mechanisms of myelination and remyelination in the central nervous system.

In the nervous system, myelin is a complex membrane structure generated by myelinating glial cells, which ensheathes axons and facilitates fast electrical ...

Primary Culture of Neurons Isolated from Embryonic Mouse Cerebellum

The use of primary cell cultures has become one of the major tools to study the nervous system in vitro. The ultimate goal of using this simplified model system is to provide a controlled microenvironment and maintain the high survival rate and the natural features of dissociated neuronal and nonneuronal cells as much as possible under in vitro conditions. In this article, we demonstrate a method of isolating primary neurons from the developing mouse cerebellum, placing them in an in vitro environment, establishing their growth, and monitoring their viability and differentiation for several weeks. This method is applicable to embryonic neurons dissociated from cerebellum between embryonic days 12&ndash;18.

Conducting in vitro experiments to reflect in vivo conditions as adequately as possible is not an easy task. The use of primary cell cultures is an important step toward understanding cell biology in a whole organism. The provided protocol outlines how to successfully grow and culture embryonic mouse cerebellar neurons.

The use of primary cell cultures has become one of the major tools to study the nervous system in vitro. The ultimate goal of using this simplified model ...

Dissection and Isolation of Murine Glia from Multiple Central Nervous System Regions

The methods presented here demonstrate laboratory procedures for the dissection of four different regions of the central nervous system (CNS) from murine neonates for the isolation of glial subpopulations. The purpose of the procedure is to dissociate microglia, oligodendrocyte progenitor cells (OPCs), and astrocytes from cortical, cerebellar, brainstem, and spinal cord tissue to facilitate further in vitro analysis. The CNS region isolation procedures allow for the determination of regional heterogeneity among glia in multiple cell culture systems. Rapid CNS region isolation is performed, followed by the mechanical removal of meninges to prevent meningeal cell contamination of glia. This protocol combines gentle tissue dissociation and plating on a specified matrix designed to preserve cell integrity and adherence. Isolating mixed glia from multiple CNS regions provides a comprehensive analysis of potentially heterogenous glia while maximizing the use of individual experimental animals. Additionally, following dissociation of regional tissue, mixed glia are further divided into multiple cell types including microglia, OPCs, and astrocytes for use in either single cell type, cell culture plate inserts, or co-culture systems. Overall, the demonstrated techniques provide a comprehensive protocol of broad applicability for careful dissection of four individual CNS regions from murine neonates and includes methods for the isolation of three individual glia cell types to examine regional heterogeneity in any number of in vitro cell culture systems or assays.

Here we present a protocol for in vitro isolation of multiple glial cell populations from a mouse CNS. This method allows for the segregation of regional microglia, oligodendrocyte precursor cells, and astrocytes to study the phenotypes of each in a variety of culture systems.

The methods presented here demonstrate laboratory procedures for the dissection of four different regions of the central nervous system (CNS) from murine ...

Cerebellar Regional Dissection for Molecular Analysis

Cerebellum plays an important role in several key functions including control of movement, balance, cognition, reward, and affect. Imaging studies indicate that distinct cerebellar regions contribute to these different functions. Molecular studies examining regional cerebellar differences are lagging as they are mostly done on whole cerebellar extracts thereby masking any distinctions across specific cerebellar regions. Here we describe a technique to reproducibly and quickly dissect four different cerebellar regions: the deep cerebellar nuclei (DCN), anterior and posterior vermal cerebellar cortex, and the cerebellar cortex of the hemispheres. Dissecting out these distinct regions allows for the exploration of molecular mechanisms that may underlie their unique contributions to balance, movement, affect and cognition. This technique may also be used to explore differences in pathological susceptibility of these specific regions across various mouse disease models.

Different cerebellar regions have been implicated to play a role in distinct behavioral outputs, yet the underlying molecular mechanisms remain unknown. This work describes a method to reproducibly and quickly dissect cerebellar cortex of the hemispheres, anterior and posterior regions of the vermis, and the deep cerebellar nuclei in order to probe for molecular differences by isolating RNA and testing for differences in gene expression.&#160;

Cerebellum plays an important role in several key functions including control of movement, balance, cognition, reward, and affect. Imaging studies ...

Efficient and Cost Effective Electroporation Method to Study Primary Cilium-Dependent Signaling Pathways in the Granule Cell Precursor

The primary cilium is a critical signaling organelle found on nearly every cell that transduces Hedgehog (Hh) signaling stimuli from the cell surface. In the granule cell precursor (GCP), the primary cilium acts as a pivotal signaling center that orchestrates precursor cell proliferation by modulating the Hh signaling pathway. The investigation of primary cilium-dependent Hh signaling machinery is facilitated by in vitro genetic manipulation of the pathway components to visualize their dynamic localization to the primary cilium. However, transfection of transgenes in the primary cultures of GCPs using the currently known electroporation methods is generally costly and often results in low cell viability and undesirable transfection efficiency. This paper introduces an efficient, cost-effective, and simple electroporation protocol that demonstrates a high transfection efficiency of ~80-90% and optimal cell viability. This is a simple, reproducible, and efficient genetic modification method that is applicable to the study of the primary cilium-dependent Hedgehog signaling pathway in primary GCP cultures.

Here, we present a reproducible in vitro electroporation protocol for genetic manipulation of primary cerebellar granule cell precursors (GCPs) that is cost-effective, efficient, and viable. Moreover, this protocol also demonstrates a straightforward method for the molecular study of primary cilium-dependent Hedgehog signaling pathways in primary GCP cells.

The primary cilium is a critical signaling organelle found on nearly every cell that transduces Hedgehog (Hh) signaling stimuli from the cell surface. In ...

Isolation and Culture of Mouse Primary Cerebellar Granule Neurons

Source: Laaper, M., et al. <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/55871">Modeling Neuronal Death and Degeneration in Mouse Primary Cerebellar Granule Neurons.</a> J. Vis. Exp. (2017)
This video demonstrates the technique for isolating and culturing primary mouse cerebellar granule neurons. Cells are treated with an antimetabolite to eliminate proliferating glial cells and produce a pure culture of cerebellar granule neurons.

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Experimental Preparation
NOTE: ...

JoVE Encyclopedia of Experiments

Neuronal Culture Techniques

Primary Neuronal Culture

Isolation of Stem Cells from Postnatal Mouse Cerebellum and Differentiation into Neural Cells

Source: Edamakanti, C. R., et al. <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/60554">Purification of Prominin-1+ Stem Cells from Postnatal Mouse Cerebellum.</a> J. Vis. Exp. (2020).
This video demonstrates a method for isolating stem cells from the postnatal mouse cerebellum and differentiating them into neural cells. Stem cells from the isolated cell suspension are purified using antibody-coated magnetic beads and are cultured to form neurospheres. The neurospheres are then maintained in a differentiation medium to generate neural cells.

1. Preparation of Solutions

	Prepare tissue dissociation solution made from sterile phenol red-containing Dulbecco&#39;s phosphate-buffered saline (DPBS) ...

Isolation of Distinct Cell Populations from the Developing Cerebellum by Microdissection

Summary

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Chapters in this video

Ex Vivo Culture of Chick Cerebellar Slices and Spatially Targeted Electroporation of Granule Cell Precursors

Isolation and Culture of Post-Natal Mouse Cerebellar Granule Neuron Progenitor Cells and Neurons

Isolation of Cortical Microglia with Preserved Immunophenotype and Functionality From Murine Neonates

Preparation and Immunostaining of Myelinating Organotypic Cerebellar Slice Cultures

Primary Culture of Neurons Isolated from Embryonic Mouse Cerebellum

Dissection and Isolation of Murine Glia from Multiple Central Nervous System Regions

Cerebellar Regional Dissection for Molecular Analysis

Efficient and Cost Effective Electroporation Method to Study Primary Cilium-Dependent Signaling Pathways in the Granule Cell Precursor

Isolation and Culture of Mouse Primary Cerebellar Granule Neurons

Isolation of Stem Cells from Postnatal Mouse Cerebellum and Differentiation into Neural Cells