Name: lectin-based isolation and culture of mouse embryonic motoneurons
Uploaded: 2011-09-15T13:00:00
Duration: 9 min 25 s
Description: Watch this Scientific Journal Video about Lectin-based Isolation and Culture of Mouse Embryonic Motoneurons at JoVE.com

We thank Sandra Bargen for excellent technical support. This work was supported by the Protein research department (PRD, TP A1.2 (R.C. and T.S.), and the RUB Research Fond, Rektoratsprogramme &#x2013; wissenschaftlicher Nachwuchs (A.K.). The monoclonal antibodies 39.4D5 and F55A10 were obtained from the Developmental Studies Hybridoma Bank (DSHB, Iowa city, IA).

For special reagents: see Tab. 1. All other reagents listed are in cell culture grade quality obtained from Sigma Aldrich.
1. PORN-H/laminin coating of dishes/coverslips
<ol>
 <li>Prepare working solution of 0.5 mg/ml Poly-DL-ornithine hydrobromide (PORN-H) in 150 mM borate buffer pH 8.35.
 <ol>
 <li>A stock solution of 50 mg PORN/1 ml borate buffer can be prepared in advance and stored at -20&deg;C.</li>
 </ol>
 </li>
 <li>Sterilize glass coverslips by flaming in 100% ethanol and let them air-dry.</li>
 <li>Cover the surface with sufficient PORN-H solution overnight at 4&deg;C.</li>
 <li>On the next day wash three times with sterilized water and air-dry coverslips.</li>
 <li>Prepare working solution of 2.5 &mu;g/ml laminin in HBSS
 <ol>
 <li>Aliquots can be stored at -20&deg;C. Thaw at 4&deg;C immediately before use.</li>
 </ol>
 </li>
 <li>Cover the surface of the PORN-H-coated coverslips with the laminin solution and let them incubate for at least 2 h at room temperature until use.</li>
</ol>
2. Lectin coating of the purification plate
<ol>
 <li>Prepare a solution of 10 mM Tris in sterilized water, pH 9.5.</li>
 <li>Add lectin (resolve the lectin (Sigma L9640) powder in HBSS) to a final concentration of 10 &mu;g/ml.</li>
 <li>Coat the surface of a 10 cm cell culture dish with 8 ml of the lectin solution and let it incubate for at least 30 min at room temperature.</li>
 <li>Wash the plate three times with HBSS and store in HBSS until use.</li>
 <li>Prepare a 30mM KCl, 0.8% (w/v) NaCl depolarisation-solution. Sterilize by filtration and store at room temperature (RT).</li>
</ol>
3. Motoneuron culture medium
<ol>
 <li>Thaw horse serum overnight at 4&deg;C and inactivate at 55&deg;C for 30 min, Aliquot into 5 ml and store at -20&deg;C. Thaw aliquot directly before use at RT.</li>
 <li>Store B27-supplement in 1 ml aliqouts at -20&deg;C. Thaw B27 supplement at RT immediately before use. Avoid freeze and thaw cycles.</li>
 <li>Aliquot glutamax into 0.5 ml aliquots and store at -20&deg;C and use it at 1:100.</li>
 <li>Prepare a stock solution of 10 &mu;g/ml CNTF in sterilized water with 0.1% BSA. Store at -20&deg;C.</li>
 <li>Mix 46 ml neurobasal medium with 2.5 ml horse serum, 1 ml B27-supplement and 0.5 ml glutamax directly before use.</li>
 <li>Add CNTF to a final concentration of 10 ng/ml medium and pre-warm medium to 37&deg;C.</li>
</ol>
4. Spinal cord dissection
<ol>
 <li>Sacrifice an E12.5 pregnant mouse and remove the embryos carefully. Add enough RT HBSS to completely cover the embryos.</li>
 <li>Remove the head and tail and place the embryo back side up with straddled limbs.</li>
 <li>Fix the embryo with one forceps and remove the outer skin with the other forceps.</li>
 <li>Remove the spinal cord by piercing the forceps under it and lift it up with saw-like movements on both sides of the spinal cord.</li>
 <li>Transfer the isolated spinal cord to a new dish with HBSS and open the central channel of the spinal cord on the dorsal side.</li>
 <li>Remove the dorsal root ganglia by removing the enclosing meninges of the spinal cord.</li>
 <li>Collect the lumbar parts of the spinal cords of up to 6 embryos per lectin plate in an eppendorf reaction tube filled with 1 ml HBSS and store on ice until the preparation is done.</li>
</ol>
5. Enrichment of motoneurons by lectin-based purification
Note: Before starting the next steps, latest here prepare and prewarm the medium (see step 3.)
<ol>
 <li>Prepare a trypsin solution by solving 1 g Trypsin in 100 ml HBSS, store at -20&deg;C and thaw at 4&deg;C.</li>
 <li>Mix 1g trypsin-inhibitor with 98 ml HBSS and 2 ml of 1 M HEPES pH 7.4, 1 ml aliquots should be stored at 4&deg;C.</li>
 <li>Transfer the tube with the spinal cords to a cell culture hood and carefully remove 700 &mu;l of the HBSS.</li>
 <li>Add 7.5 &mu;l trypsin solution and mix by carefully inverting the tube.</li>
 <li>Perform trypsination for 8 min at 37&deg;C.</li>
 <li>Stop the reaction by adding 30 &mu;l trypsin inhibitor and pipette up and down (triturate) carefully 10-15 times with a 1000 &mu;l pipette tip until no more cell aggregates are visible. Repeat the trituration with a 20-200 &mu;l pipette and the corresponding tip.</li>
 <li>Pipette the cell solution into the HBSS-filled lectin plate and disperse the cells by gently rotating the plate.</li>
 <li>Keep the lectin plate for 60 min at RT on a vibration-free surface. Cover it to avoid contamination.</li>
 <li>Remove the HBSS very gently and wash the plate carefully 4 times with pre-warmed HBSS to remove cell fragments and the not attached/unattached cells.</li>
 <li>Immediately after the last washing step, add 500 &mu;l depolarisation solution to the plate and let it incubate for 1min.</li>
 <li>Facilitate the detachment of the lectin bound cells by shaking and tapping the plate.</li>
 <li>Fill up the plate with 2 ml pre-warmed culture medium and transfer to a 15 ml falcon tube.</li>
 <li>Count cell number with a neubauer counting chamber.</li>
 <li>Plate the cell on the laminin coated coverslips or plates in an appropriate number depending on the application.</li>
 <li>Perform a medium exchange on day 1 and subsequently every second day by careful replacement of 50% of the old medium. Always use pre-warmed, freshly prepared new culture medium.</li>
</ol>
6. Representative results:
Embryonic motoneurons can be obtained from mouse embryos at E12 to E14. 2-3% of the total cell population of the lumbar spinal cord consists of motoneurons and as the dorsal root ganglionic neurons are p75NTR-positive as well, it is important to get rid of these cells before starting the lectin-based preplating procedure (For overview see Figure 1 and Figure 2). Secondly, the meninges include strongly dividing cells that should be excluded as well, as they cannot be properly removed by the lectin-based preplating procedure (Figure 2 and Figure 3). In worse cases, these cells can overgrow the culture plates. The representative pictures in Figure 3B and 3D also give an impression about the lower cell density after washing procedures have been performed (see step 5.9 of the protocol). Cultures of embryonic mouse motoneurons are usually maintained for up to five or seven days. During this time period the cells establish their neurites up to a maximum length (Figure 4). Neurite lengths strongly depend on the substrate on the plates8. The typical substrate is laminin which is coated on PORN-H covered culture dishes. Motoneurons can also grow out on other or less defined substrates, like extracellular matrices generated by water lysis8. In cases of suboptimal coating, neurite length and growth cone area are decreased and cell death usually increases. Trophic support plays a crucial role for survival of embryonic mouse motoneurons in culture. Survival of initially plated cells on day seven drop to 10% to 20% without trophic support6. Brain derived neurotrophic factor (BDNF) and Ciliary neurotrophic factor (CNTF) are the most commonly used factors but others can promote survival as well [Leukemia inhibitory factor (LIF), Cardiotrophin-1 (CT-1), basic fibroblast growth factor (bFGF), Insulin-like growth factor 1 (IGF-1)9].
<img alt="Figure 1" src="/files/ftp_upload/3200/3200fig1.jpg" /> 
Figure 1. Flow scheme for the preparation of embryonic mouse motoneurons. The schematic drawing illustrates the different steps for isolation and culture of mouse embryonic motoneurons. Abbreviations: SC: spinal cord; HBSS: Hanks balanced salt solution; MN: motoneuron.
<img alt="Figure 2" src="/files/ftp_upload/3200/3200fig2.jpg" /> 
Figure 2. Isolation of the lumbar part of the spinal cord from E12.5 mouse. A: E12.5 mouse embryo. For the next step, the head and the tail are removed and the body is placed in a dorsal-up position B: Embryo in a dorsal-up position. Forceps at the left and right of the spinal cord are fixing the embryo body in its position. One of the forceps is subsequently used to cut the skin and to cut underneath the spinal cord the other is used to fix the embryo in its position. C: Isolated spinal cord from E12.5 mouse embryo. The parts of the spinal cord (cervical, thoracic, lumbar) are indicated. The spinal cord is surrounded by the meninges and part of the dorsal root ganglia still attach to them. D: The spinal cord is longitudinally cut on the dorsal side to open it towards the central canal. E: The meninges on the ventral side are now taken off from the flattened spinal cord. Note: While taken off, the lumbar part is marked by a small cut. F: Only the lumbar part of the spinal cord is then taken for further procedures.
<img alt="Figure 3" src="/files/ftp_upload/3200/3200fig3.jpg" /> 
Figure 3. Determination and isolation of Islet-positive cells from the lumbar spinal cord of E12.5 mouse embryos. A: The Islet-1/2 antibody labels motoneuron columns within the embryonic lumbar spinal cord (red, arrow) and Hoechst labels all nuclei within the section. Cross section of an E12.5 lumar spinal cord. E12.5 mouse embryos were immersion-fixed in 4% paraformaldehyde for 6 h, washed 3 times with phosphate-buffered saline and treated with sucrose according to standard procedures. Cryosections of 20 &mu;m were taken with a cryostat and the sections were handled for immunhistochemical staining according to standard procedures11. The sections ware subsequently labeled with Hoechst for localization of the cell nuclei. Note that the dorsal root ganglionic neurons are Islet-1/2-positive as well and that the preparation procedure excludes this tissue parts from the isolation procedure. B: Islet-1/2 labeled (red, arrow) dissociated lumbar spinal cord cells from E12.5 embryos, left - before and right - after lectin-based preplating. The cells were counterstained with Hoechst to visualize all cell nuclei. C: Quantification of Islet-1/2-positive cells before and after lectin-based preplating. D: Nkx6.1 labeled motoneurons from E12.5 embryos after one day in culture. The cells were counterstained with Hoechst to cisualieze all nuclei. Note that 90% of all cells are Nkx6.1 positive. Abbreviations: SC: spinal cord; MN: motoneuron; DRG: dorsal root ganglion.
<img alt="Figure 4" src="/files/ftp_upload/3200/3200fig4.jpg" /> 
Figure 4. Characterisation of E12.5 mouse motoneurons. A and B: Enriched isolated E12.5 mouse lumbar spinal motoneurons express p75NTR at 0 days in vitro (0div) and on day 2 in vitro (2div). Cells were fixed with 4% paraformaldehyde and subsequently stained for p75NTR according to standard procedures. Cells were counterstained using Hoechst to visualize all nuclei. C: Enriched motoneurons after 5 days in vitro (5div) on PORN-H and laminin as culture substrates and in the presence of CNTF stained for &beta;-III-tubulin. Note that the cells have grown out long neurites and display the typical motoneuron morphology with one longer (branched) process and one or more shorter processes (axons and dendrites). Abbreviations: MN: motoneuron; div: days in vitro.
<table border="1">
 <tbody>
 <tr>
 <td rowspan="2"> </td>
 <td colspan="3">After dissociation without Lectin-based preplating</td>
 </tr>
 <tr>
 <td>Total number of cells/SC</td>
 <td>Trypan blue-positive cells [%]</td>
 <td>Islet-1/2- positive cells [%]</td>
 </tr>
 <tr>
 <td>1</td>
 <td>995.0</td>
 <td>13,1</td>
 <td>9,5</td>
 </tr>
 <tr>
 <td>2</td>
 <td>889.4</td>
 <td>8,0</td>
 <td>10,0</td>
 </tr>
 <tr>
 <td>3</td>
 <td>1.180.0</td>
 <td>11,0</td>
 <td>10,8</td>
 </tr>
 <tr>
 <td>Mean &plusmn; SD</td>
 <td>1.021.0 &plusmn; 147.1</td>
 <td>10,7 &plusmn; 2,6</td>
 <td>10,1 &plusmn; 0,7</td>
 </tr>
 <tr>
 <td colspan="4"> </td>
 </tr>
 <tr>
 <td rowspan="2"> </td>
 <td>With lectin-based preplating</td>
 </tr>
 <tr>
 <td>Number of cells after preplating/SC</td>
 <td>Trypan blue-positive cells [%]</td>
 <td>Islet-1/2- positive cells [%]</td>
 </tr>
 <tr>
 <td>1</td>
 <td>189.6</td>
 <td>9,9</td>
 <td>70,5</td>
 </tr>
 <tr>
 <td>2</td>
 <td>168.8</td>
 <td>3,7</td>
 <td>76,1</td>
 </tr>
 <tr>
 <td>3</td>
 <td>125.0</td>
 <td>0,0</td>
 <td>72,5</td>
 </tr>
 <tr>
 <td>Mean &plusmn; SD</td>
 <td>161.1 &plusmn; 33.0</td>
 <td>4,5 &plusmn; 5,0</td>
 <td>73,0 &plusmn; 2,8</td>
 </tr>
 </tbody>
</table>
Table 1. Summary of representative isolation procedures for mouse embryonic motoneurons from stage E12.5. Results from 3 different isolation procedures especially for this purpose are given as total numbers or percentage numbers &plusmn; SD. Abbreviation: SC: spinal cord; SD: standard deviation.
<table border="1">
 <tbody>
 <tr>
 <td>Islet-1/2 positive cells with p75 panning [%]</td>
 <td>Islet-1/2 positive cells with Lectin-based panning [%]</td>
 </tr>
 <tr>
 <td>92,0 &plusmn; 3,5 1</td>
 <td>73,0 &plusmn; 2,8</td>
 </tr>
 </tbody>
</table>
Table 2. Comparison of Lectin-based and p75-based1 panning motoneuron cell numbers. Results are given as percentage numbers &plusmn; SD.

<ol>
	<li>Vallstedt, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Different+levels+of+repressor+activity+assign+redundant+and+specific+roles+to+Nkx6+genes+in+motor+neuron+and+interneuron+specification.">Different levels of repressor activity assign redundant and specific roles to Nkx6 genes in motor neuron and interneuron specification.</a> Neuron. 31, 743-755 (2001).</li><li>Tsuchida, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Topographic+organization+of+embryonic+motor+neurons+defined+by+expression+of+LIM+homeobox+genes.">Topographic organization of embryonic motor neurons defined by expression of LIM homeobox genes.</a> Cell. 79, 957-970 (1994).</li><li>Lin, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functionally+related+motor+neuron+pool+and+muscle+sensory+afferent+subtypes+defined+by+coordinate+ETS+gene+expression.">Functionally related motor neuron pool and muscle sensory afferent subtypes defined by coordinate ETS gene expression.</a> Cell. 95, 393-407 (1998).</li><li>Wiese, S., Beck, M., Karch, C., Sendtner, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signalling+mechanisms+for+survival+of+lesioned+motoneurons.">Signalling mechanisms for survival of lesioned motoneurons.</a> Acta Neurochir. , 21-35 (2004).</li><li>Gavins, F. N., Chatterjee, B. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravital+microscopy+for+the+study+of+mouse+microcirculation+in+anti-inflammatory+drug+research:+focus+on+the+mesentery+and+cremaster+preparations.">Intravital microscopy for the study of mouse microcirculation in anti-inflammatory drug research: focus on the mesentery and cremaster preparations.</a> J. Pharmacol. Toxicol. Methods. 49, 1-14 (2004).</li><li>Bullen, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microscopic+imaging+techniques+for+drug+discovery.">Microscopic imaging techniques for drug discovery.</a> Nat. Rev. Drug Discov. 7, 54-67 (2008).</li><li>Hazelwood, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Entering+the+Portal:+Understanding+the+Digital+Image+Recorded+Through+a+Microscope.">Entering the Portal: Understanding the Digital Image Recorded Through a Microscope.</a> Imaging Cellular and Molecular Biological Functions. , 3-43 (2007).</li><li>Hickey, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=L-selectin+facilitates+emigration+and+extravascular+locomotion+of+leukocytes+during+acute+inflammatory+responses+in+vivo.">L-selectin facilitates emigration and extravascular locomotion of leukocytes during acute inflammatory responses in vivo.</a> J. Immunol. 165, 7164-7170 (2000).</li><li>Cara, D. C., Kubes, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravital+microscopy+as+a+tool+for+studying+recruitment+and+chemotaxis.">Intravital microscopy as a tool for studying recruitment and chemotaxis.</a> Methods Mol. Biol. 239, 123-132 (2004).</li><li>Liu, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LSP1+is+an+endothelial+gatekeeper+of+leukocyte+transendothelial+migration.">LSP1 is an endothelial gatekeeper of leukocyte transendothelial migration.</a> J. Exp. Med. 201, 409-418 (2005).</li><li>Heit, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PI3K+accelerates,+but+is+not+required+for,+neutrophil+chemotaxis+to+fMLP.">PI3K accelerates, but is not required for, neutrophil chemotaxis to fMLP.</a> J. Cell Sci. 121, 205-214 (2008).</li></ol>

No conflicts of interest declared.

The advantage of this lectin-based preplating technique is that it is less expensive than the p75NTR-based panning procedure, and the lectin is more stable than antibodies. The enrichment listed in Fig. 2 and Tab. 1 shows that the procedure allows to the purification of similar numbers of cells and that a majority of these cells express the motoneuron marker Islet-1/2. The most critical step is the isolation procedure for the lumbar spinal cord. Removing the meninges and the DRGs (Fig. 2) is essential for the following purification procedure by lectin-based preplating. If this has been managed correctly, almost all cells express the p75NTR (for representative picture see Fig. 3a). The reason for the difference between expression of Islet-1/2 and p75NTR is most probably because lumbar motoneurons differentially express higher and lower levels of Islet-1/210. In case of low levels of Islet-1/2 expression this might have escaped our attention in the immuncytochemical staining and subsequent counting as we were stringent with respect to positive versus negative cells (Tab. 1). Additionally, Table 1 clearly shows that the isolated cells survive the procedure in a healthy condition, as there are only few trypan-blue positive cells that indicate that the cells are irreversibly damaged. This alterative procedure also allows isolation of motoneurons from single embryos and therefore of mixed genotype litters from embryonic mice. In conclusion, this alternative to the panning procedure with the p75NTR antibody6 has similar if not identical capacities in terms of possible application ranges and provides a cheap and efficient alternative purification method for mouse embryonic motoneurons.

<table border="1">
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Poly-DL-ornithine hydrobromide</td>
			<td>Sigma </td>
			<td>P8638</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Laminin</td>
			<td>Invitrogen</td>
			<td>23017-015</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>HBSS</td>
			<td>Gibco</td>
			<td>14170</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Glass coverslips</td>
			<td>Thermo </td>
			<td>&nbsp;</td>
			<td>&Oslash; 10 or 14mm</td>
		</tr>
		<tr>
			<td>Lectin</td>
			<td>Sigma</td>
			<td>L5142</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Cell culture dish</td>
			<td>Nunc</td>
			<td>150350</td>
			<td>Nunclon delta surface</td>
		</tr>
		<tr>
			<td>Horse serum</td>
			<td>Linaris</td>
			<td>SHD3250ZK</td>
			<td>Each batch has to be tested for MN culture</td>
		</tr>
		<tr>
			<td>B27 Supplement</td>
			<td>Gibco</td>
			<td>17504-044</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Glutamax</td>
			<td>Gibco</td>
			<td>35050-038</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>CNTF</td>
			<td>Sigma</td>
			<td>N0513</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>BSA </td>
			<td>Applichem</td>
			<td>A1391</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Neurobasal </td>
			<td>Gibco</td>
			<td>21103-041</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Stainless steel, size 4 or 5</td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Worthington</td>
			<td>LS003707</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Trypsin-Inhibitor</td>
			<td>Sigma</td>
			<td>T6522</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Anti-Islet-1</td>
			<td>DSHB</td>
			<td>39.4D5</td>
			<td>Cell culture supernatant</td>
		</tr>
		<tr>
			<td>Anti-Nkx6.1</td>
			<td>DSHB</td>
			<td>F55A10</td>
			<td>Cell culture supernatant</td>
		</tr>
		<tr>
			<td>Anti-p75NTR</td>
			<td>Abcam</td>
			<td>Ab8874</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>
Table 3. Table of specific reagents and equipment.

lectin-based isolation and culture of mouse embryonic motoneurons

Spinal motoneurons develop towards postmitotic stages through early embryonic nervous system development and subsequently grow out dendrites and axons. Neuroepithelial cells of the neural tube that express Nkx6.1 are the unique precursor cells for spinal motoneurons1. Though postmitotic motoneurons move towards their final position and organize themselves into columns along the spinal tract2,3. More than 90% of all these differentiated and positioned motoneurons express the transcription factors Islet 1/2. They innervate the muscles of the limbs as well as those of the body and the inner organs. Among others, motoneurons typically express the high affinity receptors for brain derived neurotrophic factor (BDNF) and Neurotrophin-3 (NT-3), the tropomyosin-related kinase B and C (TrkB, TrkC). They do not express the tropomyosin-related kinase A (TrkA)4. Beside the two high affinity receptors, motoneurons do express the low affinity neurotrophin receptor p75NTR. The p75NTR can bind all neurotrophins with similar but lower affinity to all neurotrophins than the high affinity receptors would bind the mature neurotrophins. Within the embryonic spinal cord, the p75NTR is exclusively expressed by the spinal motoneurons5. This has been used to develop motoneuron isolation techniques to purify the cells from the vast majority of surrounding cells6. Isolating motoneurons with the help of specific antibodies (panning) against the extracellular domains of p75NTR has turned out to be an expensive method as the amount of antibody used for a single experiment is high due to the size of the plate used for panning. A much more economical alternative is the use of lectin. Lectin has been shown to specifically bind to p75NTR as well7. The following method describes an alternative technique using wheat germ agglutinin for a preplating procedure instead of the p75NTR antibody. The lectin is an extremely inexpensive alternative to the p75NTR antibody and the purification grades using lectin are comparable to that of the p75NTR antibody. Motoneurons from the embryonic spinal cord can be isolated by this method, survive and grow out neurites.

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Lectin-based Isolation and Culture of Mouse Embryonic Motoneurons

An alternative way of isolating mouse embryonic motoneurons from the spinal cord is described. The method takes into account the fact that lectin can bind to the low affinity nerve growth factor receptor p75NTR. This lectin-based preplating allows a purification similar to that with a specific antibody against the p75NTR.

Spinal motoneurons develop towards postmitotic stages through early embryonic nervous system development and subsequently grow out dendrites and axons. ...

lectin-based-isolation-and-culture-of-mouse-embryonic-motoneurons

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Neuroscience

Dissection and Culture of Commissural Neurons from Embryonic Spinal Cord

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies of these neurons are located in the dorsal spinal cord and their axons follow stereotyped trajectories during embryonic development. Commissural axons initially project ventrally towards the floorplate. After crossing the midline, these axons turn anteriorly and project towards the brain. Each of these steps is regulated by the action of several guidance cues. Cultures highly enriched in commissural neurons are ideally suited for many experiments addressing the mechanisms of axon pathfinding, including turning assays, immunochemistry and biochemistry. Here, we describe a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. First, the spinal cord is isolated and dorsal strips are dissected out. The dorsal tissue is then dissociated into a cell suspension by trypsinization and mechanical disruption. Neurons are plated onto poly-L-lysine-coated glass coverslips or tissue-culture dishes. After 30 hours in vitro, most neurons have extended an axon. The purity of the culture (Yam et al. 2009), typically over 90%, can be assessed by immunolabeling with the commissural neuron markers DCC, LH2 and TAG1 (Helms and Johnson, 1998). This neuronal preparation is a useful tool for in vitro studies of the cellular and molecular mechanisms of commissural axon growth and guidance during spinal cord development.

This video demonstrates a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. Dissociated commissural neurons are useful to study the cellular and molecular mechanisms of axon growth and guidance.

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies ...

Nucleofection and Primary Culture of Embryonic Mouse Hippocampal and Cortical Neurons

Hippocampal and cortical neurons have been used extensively to study central nervous system (CNS) neuronal polarization, axon/dendrite outgrowth, and synapse formation and function. An advantage of culturing these neurons is that they readily polarize, forming distinctive axons and dendrites, on a two dimensional substrate at very low densities. This property has made them extremely useful for determining many aspects of neuronal development. Furthermore, by providing glial conditioning for these neurons they will continue to develop, forming functional synaptic connections and surviving for several months in culture. In this protocol we outline a technique to dissect, culture and transfect embryonic mouse hippocampal and cortical neurons. Transfection is accomplished by electroporating DNA into the neurons before plating via nucleofection. This protocol has the advantage of expressing fluorescently-tagged fusion proteins early in development (~4-8hrs after plating) to study the dynamics and function of proteins during polarization, axon outgrowth and branching. We have also discovered that this single transfection before plating maintains fluorescently-tagged fusion protein expression at levels appropriate for imaging throughout the lifetime of the neuron (&gt; 2 months in culture). Thus, this methodology is useful for studying protein localization and function throughout CNS development with little or no disruption of neuronal function.

This protocol outlines the steps required to dissect, transfect via electroporation and culture mouse hippocampal and cortical neurons. Short-term cultures may be used for studies of axon outgrowth and guidance, while long-term cultures can be used for studies of synaptogenesis and dendritic spine analysis.

Hippocampal and cortical neurons have been used extensively to study central nervous system (CNS) neuronal polarization, axon/dendrite outgrowth, and ...

Establishing Embryonic Mouse Neural Stem Cell Culture Using the Neurosphere Assay

In mammalians, stem cells acts as a source of undifferentiated cells to maintain cell genesis and renewal in different tissues and organs during the life span of the animal. They can potentially replace cells that are lost in the aging process or in the process of injury and disease. The existence of neural stem cells (NSCs) was first described by Reynolds and Weiss (1992) in the adult mammalian central nervous system (CNS) using a novel serum&#8208;free culture system, the neurosphere assay (NSA). Using this assay, it is also feasible to isolate and expand NSCs from different regions of the embryonic CNS. These in vitro expanded NSCs are multipotent and can give rise to the three major cell types of the CNS. While the NSA seems relatively simple to perform, attention to the procedures demonstrated here is required in order to achieve reliable and consistent results. This video practically demonstrates NSA to generate and expand NSCs from embryonic day 14-mouse brain tissue and provides technical details so one can achieve reproducible neurosphere cultures. The procedure includes harvesting E14 mouse embryos, brain microdissection to harvest the ganglionic eminences, dissociation of the harvested tissue in NSC medium to gain a single cell suspension, and finally plating cells in NSA culture. After 5-7 days in culture, the resulting primary neurospheres are passaged to further expand the number of the NSCs for future experiments.

This video protocol demonstrates the application of the neurosphere assay for the isolation and expansion of neural stem cells from the ganglionic eminences of embryonic day 14-mouse brain.

In mammalians, stem cells acts as a source of undifferentiated cells to maintain cell genesis and renewal in different tissues and organs during the life ...

Isolation and Culture of Rat Embryonic Neural Cells: A Quick Protocol

We are describing a quick method to dissociate and culture hippocampal or cortical neurons from E15-17 rat embryos. The procedure can be applied successfully to the isolation of mouse and human primary neurons and neural progenitors. Dissociated neurons are maintained in serum-free medium up to several weeks. These cultures can be used for nucleofection, immunocytochemistry, nucleic acids preparation, as well as electrophysiology. Older neuronal cultures can also be transfected with a good efficiency rate by lentiviral transduction and, less efficiently, with calcium phosphate or lipid-based methods such as lipofectamine.

We describe a rapid methodology to isolate and culture hippocampal and cortical neurons from rodent embryos. This protocol allows us to perform experiments in which nearly pure neuronal cultures are required.

We are describing a quick method to dissociate and culture hippocampal or cortical neurons from E15-17 rat embryos. The procedure can be applied ...

Isolation and Culture of Neural Crest Cells from Embryonic Murine Neural Tube

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to mesenchymal transition (EMT) and migrates throughout the embryo, giving rise to diverse cell types 1-3. NC also has the unique ability to influence the differentiation and maturation of target organs4-6. When explanted in vitro, NC progenitors undergo self-renewal, migrate and differentiate into a variety of tissue types including neurons, glia, smooth muscle cells, cartilage and bone. 
NC multipotency was first described from explants of the avian neural tube7-9. In vitro isolation of NC cells facilitates the study of NC dynamics including proliferation, migration, and multipotency. Further work in the avian and rat systems demonstrated that explanted NC cells retain their NC potential when transplanted back into the embryo10-13. Because these inherent cellular properties are preserved in explanted NC progenitors, the neural tube explant assay provides an attractive option for studying the NC in vitro. 
To attain a better understanding of the mammalian NC, many methods have been employed to isolate NC populations. NC-derived progenitors can be cultured from post-migratory locations in both the embryo and adult to study the dynamics of post-migratory NC progenitors11,14-20, however isolation of NC progenitors as they emigrate from the neural tube provides optimal preservation of NC cell potential and migratory properties13,21,22. Some protocols employ fluorescence activated cell sorting (FACS) to isolate a NC population enriched for particular progenitors11,13,14,17. However, when starting with early stage embryos, cell numbers adequate for analyses are difficult to obtain with FACS, complicating the isolation of early NC populations from individual embryos. Here, we describe an approach that does not rely on FACS and results in an approximately 96% pure NC population based on a Wnt1-Cre activated lineage reporter23. 
The method presented here is adapted from protocols optimized for the culture of rat NC11,13. The advantages of this protocol compared to previous methods are that 1) the cells are not grown on a feeder layer, 2) FACS is not required to obtain a relatively pure NC population, 3) premigratory NC cells are isolated and 4) results are easily quantified. Furthermore, this protocol can be used for isolation of NC from any mutant mouse model, facilitating the study of NC characteristics with different genetic manipulations. The limitation of this approach is that the NC is removed from the context of the embryo, which is known to influence the survival, migration and differentiation of the NC2,24-28.

Isolation of embryonic neural crest from the neural tube facilitates the use of in vitro methods for studying migration, self-renewal, and multipotency of neural crest.

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to ...

Dissection, Culture, and Analysis of Xenopus laevis Embryonic Retinal Tissue

The process by which the anterior region of the neural plate gives rise to the vertebrate retina continues to be a major focus of both clinical and basic research. In addition to the obvious medical relevance for understanding and treating retinal disease, the development of the vertebrate retina continues to serve as an important and elegant model system for understanding neuronal cell type determination and differentiation1-16. The neural retina consists of six discrete cell types (ganglion, amacrine, horizontal, photoreceptors, bipolar cells, and M&uuml;ller glial cells) arranged in stereotypical layers, a pattern that is largely conserved among all vertebrates 12,14-18.
While studying the retina in the intact developing embryo is clearly required for understanding how this complex organ develops from a protrusion of the forebrain into a layered structure, there are many questions that benefit from employing approaches using primary cell culture of presumptive retinal cells 7,19-23. For example, analyzing cells from tissues removed and dissociated at different stages allows one to discern the state of specification of individual cells at different developmental stages, that is, the fate of the cells in the absence of interactions with neighboring tissues 8,19-22,24-33. Primary cell culture also allows the investigator to treat the culture with specific reagents and analyze the results on a single cell level 5,8,21,24,27-30,33-39. Xenopus laevis, a classic model system for the study of early neural development 19,27,29,31-32,40-42, serves as a particularly suitable system for retinal primary cell culture 10,38,43-45.
Presumptive retinal tissue is accessible from the earliest stages of development, immediately following neural induction 25,38,43. In addition, given that each cell in the embryo contains a supply of yolk, retinal cells can be cultured in a very simple defined media consisting of a buffered salt solution, thus removing the confounding effects of incubation or other sera-based products 10,24,44-45.
However, the isolation of the retinal tissue from surrounding tissues and the subsequent processing is challenging. Here, we present a method for the dissection and dissociation of retinal cells in Xenopus laevis that will be used to prepare primary cell cultures that will, in turn, be analyzed for calcium activity and gene expression at the resolution of single cells. While the topic presented in this paper is the analysis of spontaneous calcium transients, the technique is broadly applicable to a wide array of research questions and approaches (Figure 1).

Dissection, Culture, and Analysis of Xenopus laevis Embryonic Retinal Tissue

Xenopus laevis provides an ideal model system for studying cell fate specification and physiological function of individual retinal cells in primary cell culture. Here we present a technique for dissecting retinal tissues and generating primary cell cultures that are imaged for calcium activity and analyzed by in situ hybridization.

The process by which  the anterior region of the neural plate gives rise to the vertebrate retina  continues to be a major focus of both clinical and ...

Isolation and Culture of Mouse Cortical Astrocytes

Astrocytes are an abundant cell type in the mammalian brain, yet much remains to be learned about their molecular and functional characteristics. In vitro astrocyte cell culture systems can be used to study the biological functions of these glial cells in detail. This video protocol shows how to obtain pure astrocytes by isolation and culture of mixed cortical cells of mouse pups. The method is based on the absence of viable neurons and the separation of astrocytes, oligodendrocytes and microglia, the three main glial cell populations of the central nervous system, in culture. Representative images during the first days of culture demonstrate the presence of a mixed cell population and indicate the timepoint, when astrocytes become confluent and should be separated from microglia and oligodendrocytes. Moreover, we demonstrate purity and astrocytic morphology of cultured astrocytes using immunocytochemical stainings for well established and newly described astrocyte markers. This culture system can be easily used to obtain pure mouse astrocytes and astrocyte-conditioned medium for studying various aspects of astrocyte biology.

Astrocytes have been recognized to be versatile cells participating in fundamental biological processes that are essential for normal brain development and function, and central nervous system repair. Here we present a rapid procedure to obtain pure mouse astrocyte cultures to study the biology of this major class of central nervous system cells.

Astrocytes  are an abundant cell type in the mammalian brain, yet much remains to be  learned about their molecular and functional characteristics. In ...

Isolation and Culture of Endothelial Cells from the Embryonic Forebrain

Embryonic brain endothelial cells can serve as an important tool in the study of angiogenesis and neurovascular development and interactions. The two vascular networks of the embryonic forebrain, pial and periventricular, are spatially distinctive and have different origins and growth patterns. Endothelial cells from the pial and periventricular vascular networks have unique gene expression profiles and functions. Here we present a step-by-step protocol for isolation, culture, and verification of pure populations of endothelial cells from the periventricular vascular network (PVECs) of the embryonic forebrain (telencephalon). In this approach, telencephalon devoid of pial membrane obtained from embryonic day 15 mice is minced, digested with collagenase/dispase, and dispersed mechanically into a single cell suspension. PVECs are purified from cell suspension using positive selection with anti-CD-31/PECAM-1 antibody conjugated to MicroBeads using a strong magnetic separation method. Purified cells are cultured on collagen 1&#160;coated culture dishes in endothelial cell culture medium until they become confluent and further subcultured. PVECs obtained with this protocol exhibit cobblestone and spindle shaped phenotypes, as visualized by phase-contrast light microscopy and fluorescence microscopy. Purity of PVEC cultures was established with endothelial cell markers. In our hands, this method reliably and consistently yields pure populations of PVECs. This protocol will benefit studies aimed at gaining mechanistic insights into forebrain angiogenesis, understanding PVEC interactions, and cross-talks with neuronal cell types and holds tremendous potential for therapeutic angiogenesis.

This video demonstrates an easy and reliable strategy for preparation of pure cultures of endothelial cells from the embryonic forebrain within 10-12 days and will be useful for research focused on many aspects of cerebral angiogenesis.

Embryonic brain endothelial cells can serve as an important tool in the study of angiogenesis and neurovascular development and interactions. The two ...

Isolation, Culture and Long-Term Maintenance of Primary Mesencephalic Dopaminergic Neurons From Embryonic Rodent Brains

Degeneration of mesencephalic dopaminergic (mesDA) neurons is the pathological hallmark of Parkinson&#8217;s diseae. Study of the biological processes involved in physiological functions and vulnerability and death of these neurons is imparative to understanding the underlying causes and unraveling the cure for this common neurodegenerative disorder. Primary cultures of mesDA neurons provide a tool for investigation of the molecular, biochemical and electrophysiological properties, in order to understand the development, long-term survival and degeneration of these neurons during the course of disease. Here we present a detailed method for the isolation, culturing and maintenance of midbrain dopaminergic neurons from E12.5 mouse (or E14.5 rat) embryos. Optimized cell culture conditions in this protocol result in presence of axonal and dendritic projections, synaptic connections and other neuronal morphological properties, which make the cultures suitable for study of the physiological, cell biological and molecular characteristics of this neuronal population.

The causes of degeneration of midbrain dopaminergic neurons during Parkinson&#8217;s disease are not fully understood. Cellular culture systems provide an essential tool for study of the neurophysiological properties of these neurons. Here we present an optimized protocol, which can be utilized for in vitro modeling of neurodegeneration.

Degeneration of mesencephalic dopaminergic (mesDA) neurons is the pathological hallmark of Parkinson&#8217;s diseae. Study of the biological processes ...

Isolation and Culture of Adult Neural Stem Cells from the Mouse Subcallosal Zone

Adult neural stem cells (aNSCs) can be used for the regeneration of damaged brain tissue. NSCs have the potential for differentiation and proliferation into three types of cells: neurons, astrocytes, and oligodendrocytes. Identifying aNSC-derived regions and characterizing the aNSC properties are critical for the potential use of aNSCs and for the elucidation of their role in neural regeneration. The subcallosal zone (SCZ), located between white matter and the hippocampus, has recently been reported to contain aNSCs and continuously give rise to neuroblasts. A low percentage of aNSCs from the SCZ is differentiated into neurons; most cells are differentiated into glial cells, such as oligodendrocytes and astrocytes. These cells are suggested to have a therapeutic potential for traumatic cortical injury. This protocol describes in detail the process to generate SCZ-aNSCs from an adult mouse brain. A brain matrix with intervals of 1 mm is used to obtain the SCZ-containing coronal slices and to precisely dissect the SCZ from the whole brain. The SCZ sections are initially subjected to a neurosphere culture. A well-developed culture system allows for the verification of their characteristics and can increase research on NSCs. A neurosphere culture system provides a useful tool for determining proliferation and collecting the genuine NSCs. A monolayer culture is also an in vitro system to assay proliferation and differentiation. Significantly, this culture system provides a more homogenous environment for NSCs than the neurosphere culture system. Thus, using a discrete brain region, these culture systems will be helpful for expanding our knowledge about aNSCs and their applications for therapeutic uses.

Establishing culture systems for the expansion of adult neural stem cells (aNSCs) allows for the examination and application of aNSCs for therapy. The subcallosal zone (SCZ) has recently been recognized as a novel neuroblast-forming region in adult mice. Here, methods for the isolation, expansion, and differentiation of SCZ-aNSCs are described.

Adult neural stem cells (aNSCs) can be used for the regeneration of damaged brain tissue. NSCs have the potential for differentiation and proliferation ...