This work was supported by Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (23700412, 25122707 and 26670090 to S.Y.).

Procedures involving animal subjects have been approved by the Institutional Animal Care and Use Committee at Hamamatsu University School of Medicine.
1. Preparation of Matrices
<ol>
	<li>Boil 4-8 silicone matrices in a microwave or on a hot plate for 5 min and allow them to dry completely for 1 h under laminar flow (striped side up). 
	Note: The following procedures should be performed under laminar flow.</li>
	<li>Blow compressed air or use transparent sticky tape to remove any dust from the striped side ...

<ol>
	<li>Dickson, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+mechanisms+of+axon+guidance.">Molecular mechanisms of axon guidance.</a> Science. 298 (5600), 1959-1964 (2002).</li><li>Bashaw, G. J., Klein, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signaling+from+axon+guidance+receptors.">Signaling from axon guidance receptors.</a> Cold Spring Harb Perspect Biol. 2 (5), 001941 (2010).</li><li>Hong, K., Nishiyama, M., Henley, J., Tessier-Lavigne, M., Poo, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+signalling+in+the+guidance+of+nerve+growth+by+netrin-1.">Calcium signalling in the guidance of nerve growth by netrin-1.</a> Nature. 403 (6765), 93-98 (2000).</li><li>Ming, G. -. l., Henley, J., Tessier-Lavigne, M., Song, H. -. j., Poo, M. -. m. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrical+Activity+Modulates+Growth+Cone+Guidance+by+Diffusible+Factors.">Electrical Activity Modulates Growth Cone Guidance by Diffusible Factors.</a> Neuron. 29 (2), 441-452 (2001).</li><li>Dudanova, I., 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=Genetic+evidence+for+a+contribution+of+EphA:ephrinA+reverse+signaling+to+motor+axon+guidance.">Genetic evidence for a contribution of EphA:ephrinA reverse signaling to motor axon guidance.</a> J Neurosci. 32 (15), 5209-5215 (2012).</li><li>Ye, B. Q., Geng, Z. H., Ma, L., Geng, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Slit2+regulates+attractive+eosinophil+and+repulsive+neutrophil+chemotaxis+through+differential+srGAP1+expression+during+lung+inflammation.">Slit2 regulates attractive eosinophil and repulsive neutrophil chemotaxis through differential srGAP1 expression during lung inflammation.</a> J Immunol. 185 (10), 6294-6305 (2010).</li><li>Ly, 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=DSCAM+is+a+netrin+receptor+that+collaborates+with+DCC+in+mediating+turning+responses+to+netrin-1.">DSCAM is a netrin receptor that collaborates with DCC in mediating turning responses to netrin-1.</a> Cell. 133 (7), 1241-1254 (2008).</li><li>Hata, K., Kaibuchi, K., Inagaki, S., Yamashita, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unc5B+associates+with+LARG+to+mediate+the+action+of+repulsive+guidance+molecule.">Unc5B associates with LARG to mediate the action of repulsive guidance molecule.</a> J Cell Biol. 184 (5), 737-750 (2009).</li><li>Egea, 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=Regulation+of+EphA+4+kinase+activity+is+required+for+a+subset+of+axon+guidance+decisions+suggesting+a+key+role+for+receptor+clustering+in+Eph+function.">Regulation of EphA 4 kinase activity is required for a subset of axon guidance decisions suggesting a key role for receptor clustering in Eph function.</a> Neuron. 47 (4), 515-528 (2005).</li><li>von Philipsborn, A. C., Lang, S., Jiang, Z., Bonhoeffer, F., Bastmeyer, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Substrate-Bound+Protein+Gradients+for+Cell+Culture+Fabricated+by+Microfluidic+Networks+and+Microcontact+Printing.">Substrate-Bound Protein Gradients for Cell Culture Fabricated by Microfluidic Networks and Microcontact Printing.</a> Sci Signal. , (2007).</li><li>Jackman, R., Wilbur, J., Whitesides, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fabrication+of+submicrometer+features+on+curved+substrates+by+microcontact+printing.">Fabrication of submicrometer features on curved substrates by microcontact printing.</a> Science. 269 (5224), 664-666 (1995).</li><li>Mrksich, M., Whitesides, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+self-assembled+monolayers+to+understand+the+interactions+of+man-made+surfaces+with+proteins+and+cells.">Using self-assembled monolayers to understand the interactions of man-made surfaces with proteins and cells.</a> Annu Rev Biophys Biomol Struct. 25, 55-78 (1996).</li><li>Walter, J., Kern-Veits, B., Huf, J., Stolze, B., Bonhoeffer, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recognition+of+position-specific+properties+of+tectai+cell+membranes+by+retinal+axons+in+vitro.">Recognition of position-specific properties of tectai cell membranes by retinal axons in vitro.</a> Development. 101, 685-696 (1987).</li><li>Knoll, B., Weinl, C., Nordheim, A., Bonhoeffer, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stripe+assay+to+examine+axonal+guidance+and+cell+migration.">Stripe assay to examine axonal guidance and cell migration.</a> Nat Protoc. 2 (5), 1216-1224 (2007).</li><li>Weschenfelder, M., Weth, F., Knoll, B., Bastmeyer, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+stripe+assay:+studying+growth+preference+and+axon+guidance+on+binary+choice+substrates+in+vitro.">The stripe assay: studying growth preference and axon guidance on binary choice substrates in vitro.</a> Methods Mol Biol. 1018, 229-246 (2013).</li><li>Seiradake, E., 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=Structure+and+functional+relevance+of+the+Slit2+homodimerization+domain.">Structure and functional relevance of the Slit2 homodimerization domain.</a> EMBO Rep. 10 (7), 736-741 (2009).</li><li>Gebhardt, C., Bastmeyer, M., Weth, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Balancing+of+ephrin/Eph+forward+and+reverse+signaling+as+the+driving+force+of+adaptive+topographic+mapping.">Balancing of ephrin/Eph forward and reverse signaling as the driving force of adaptive topographic mapping.</a> Development. 139 (2), 335-345 (2012).</li><li>Atapattu, L., 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=Antibodies+binding+the+ADAM10+substrate+recognition+domain+inhibit+Eph+function.">Antibodies binding the ADAM10 substrate recognition domain inhibit Eph function.</a> J Cell Sci. 125, 6084-6093 (2012).</li><li>Stark, D. A., Karvas, R. M., Siegel, A. L., Cornelison, D. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Eph/ephrin+interactions+modulate+muscle+satellite+cell+motility+and+patterning.">Eph/ephrin interactions modulate muscle satellite cell motility and patterning.</a> Development. 138 (24), 5279-5289 (2011).</li><li>Seiradake, E., 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=FLRT+Structure:+Balancing+Repulsion+and+Cell+Adhesion+in+Cortical+and+Vascular+Development.">FLRT Structure: Balancing Repulsion and Cell Adhesion in Cortical and Vascular Development.</a> Neuron. 84 (2), 370-385 (2014).</li><li>Yamagishi, S., 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=FLRT2+and+FLRT3+act+as+repulsive+guidance+cues+for+Unc5-positive+neurons.">FLRT2 and FLRT3 act as repulsive guidance cues for Unc5-positive neurons.</a> EMBO J. 30 (14), 2920-2933 (2011).</li></ol>

The authors have nothing to disclose.

This protocol describes a stripe assay that uses recombinant protein and dissociated neurons from the E15.5 mouse hippocampus. This assay allows the efficient observation of repulsive, attractive, or neutral responses of neurons to a recombinant protein of interest placed in a striped pattern. A major advantage of this protocol is the simple method for generating the stripes, in which the protein is directly printed onto the surface of a plastic dish, compared to the traditional method, which requires special matrices, a...

Axon guidance is the process by which newly formed neurons send axons to their target during development of the nervous system1,2. Developing axons carry a highly motile structure at their tip called the growth cone. The growth cone senses extracellular cues to navigate the axon&#39;s path. Guidance molecules, such as slit, semaphorin, and ephrins, can attract or repel axons depending on their interaction with suitable receptors and co-receptors on the axon1,3,4. The activated receptors transfer signals to the growth cone that affect its cytoskeletal organization for axon and growth-cone movements.
Various methods have been developed to evaluate the action of attractant and repellent molecules. Chemo-attractants and repellents can be administered into the growth/culture medium with a gradient concentration (e.g., Dunn&#39;s chamber or &#181;-slides)5,6, in a highly concentrated spot by micro-pipette (e.g., turning assay)7 or at a homogenous concentration by bath application (e.g., growth cone collapse assay)8,9.
Other methods include a stripe assay or microcontact printing (&#181;CP), in which a chemo-attractant or repellent is coated on the surface of a plate as a substrate10-12. Thestripe assay was originally developed by Bonhoeffer and colleagues in 1987 to analyze topographical mapping in the chick retino-tectal system13. The original method required a vacuum system to coat proteins onto polycarbonate nucleopore membranes using striped and meshed matrices. In later versions, the recombinant proteins were directly printed on the surface of a culture plate in a striped pattern using narrow slit silicon matrices14,15. Recently, various research groups have successfully applied this stripe assay to the analysis of axon guidance molecule activities16-21.
Here, we present the detailed protocol for a stripe assay that measures the attraction or repulsion of axon guidance molecules for dissociated hippocampal neurons. Notably, this method can be applied in minimally equipped laboratory settings. For this assay, alternating stripes of a fluorescently labeled substrate and a control protein are generated on a plastic dish using a silicon matrix with 90-&#181;m slits and coated with laminin. In our demonstration, dissociated hippocampal neurons from E15.5 mice were cultured on alternating stripes of recombinant ectodomain of fibronectin and leucine-rich transmembrane protein-2 (FLRT2) and control Fc protein21. After 24 h of culture, both the axons and cell bodies of the neurons were strongly repelled from the FLRT2 stripes. Staining with an anti-Tau1 antibody revealed that ~90% of the neurons were distributed on the Fc-coated regions, compared to ~10% on the FLRT2-Fc, indicating that FLRT2 has a strong repulsive function for hippocampal neurons21.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>15 mL centrifuge tube</td>
			<td>Violamo&#160;</td>
			<td>1-3500-01</td>
			<td></td>
		</tr>
		<tr>
			<td>4% Paraformaldehyde (PFA)</td>
			<td>Nacalai</td>
			<td>01954-85</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 Goat anti-human IgG antibody</td>
			<td>Thermo Scientific</td>
			<td>A11013</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 594 Donkey anti-mouse IgG antibody</td>
			<td>Thermo Scientific</td>
			<td>A-21203</td>
			<td>Dilution 1/500</td>
		</tr>
		<tr>
			<td>Anti-Tau1 antibody</td>
			<td>Chemicon</td>
			<td>MAB3420</td>
			<td>Dilution 1/200</td>
		</tr>
		<tr>
			<td>Antifade</td>
			<td>Thermo Scientific</td>
			<td>P7481</td>
			<td>Alternative mounting media may be used</td>
		</tr>
		<tr>
			<td>B27 supplement</td>
			<td>Thermo Scientific</td>
			<td>17504-044</td>
			<td>Dilution 1/50</td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>Sigma</td>
			<td>01-2030-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell strainer 100 um</td>
			<td>BD Falcon</td>
			<td>352360</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifugation machine</td>
			<td>Kubota</td>
			<td>2410</td>
			<td></td>
		</tr>
		<tr>
			<td>Cover glass 18mmx18mm</td>
			<td>Matsunami</td>
			<td>18x18 mm No. 1</td>
			<td></td>
		</tr>
		<tr>
			<td>DAKO pen</td>
			<td>DAKO</td>
			<td>S2002</td>
			<td>Alternative water-repellent pen may be used</td>
		</tr>
		<tr>
			<td>Disposable scalpel</td>
			<td>Feather</td>
			<td>2975#11</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Thermo Scientific</td>
			<td>10437-028</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorecent microscope</td>
			<td>Nikon</td>
			<td>E600</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps No. 5</td>
			<td>Fine Science Tools</td>
			<td>11254-20</td>
			<td></td>
		</tr>
		<tr>
			<td>GlutaMAX</td>
			<td>Thermo Scientific</td>
			<td>35050-061</td>
			<td>Dilution 1/200</td>
		</tr>
		<tr>
			<td>Hamilton Syringe</td>
			<td>Hamilton</td>
			<td>805N</td>
			<td>22 gauge, 50 uL</td>
		</tr>
		<tr>
			<td>HBSS</td>
			<td>Thermo Scientific</td>
			<td>14170-112</td>
			<td></td>
		</tr>
		<tr>
			<td>Human IgG, Fc Fragment</td>
			<td>Jackson</td>
			<td>009-000-008</td>
			<td></td>
		</tr>
		<tr>
			<td>Laminin</td>
			<td>Thermo Scientific</td>
			<td>23017-015</td>
			<td></td>
		</tr>
		<tr>
			<td>Neurobasal</td>
			<td>Thermo Scientific</td>
			<td>21103-049</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal Donkey Serum</td>
			<td>Jackson</td>
			<td>017-000-121</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Nacalai</td>
			<td>14249-24</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Thermo Scientific</td>
			<td>15070-063</td>
			<td>Dilution 1/100</td>
		</tr>
		<tr>
			<td>Plastic culture dish, 60 mm</td>
			<td>Thermo Scientific</td>
			<td>150288</td>
			<td></td>
		</tr>
		<tr>
			<td>Silicone Matrices</td>
			<td></td>
			<td></td>
			<td>Available and purchasable from Prof. Martin Bastmeyer (bastmeyer@kit.edu)</td>
		</tr>
		<tr>
			<td>Stereo Microscope</td>
			<td>Olympus</td>
			<td>SZ61</td>
			<td></td>
		</tr>
		<tr>
			<td>Tip, 1000 uL</td>
			<td>Watson</td>
			<td>125-1000S</td>
			<td></td>
		</tr>
		<tr>
			<td>Transparent sticky tape</td>
			<td>Tesa</td>
			<td>57315</td>
			<td>Alternative sticky tape may be used</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma</td>
			<td>T8787</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan blue, 0.4%</td>
			<td>Bio-Rad</td>
			<td>145-0013</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin/EDTA</td>
			<td>Thermo Scientific</td>
			<td>25300-054</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Culture medium</td>
			<td></td>
			<td></td>
			<td>Neurobasal supplemented with B27, GlutaMAX and Penicillin-Streptomycin.</td>
		</tr>
	</tbody>
</table>

stripe assay to study the attractive or repulsive activity of a protein substrate using dissociated hippocampal neurons

Growing axons develop a highly motile structure at their tip, termed the growth cone. The growth cone contacts extracellular environmental cues to navigate axonal growth. Netrin, slit, semaphorin, and ephrins are known guidance molecules that can attract or repel axons upon binding to receptors and co-receptors on the axon. The activated receptors initiate various signaling molecules in the growth cone that alter the structure and movement of the neuron. Here, we describe the detailed protocol for a stripe assay to assess the ability of a guidance molecule to attract or repel neurons. In this method, dissociated hippocampal neurons from E15.5 mice are cultured on laminin-coated dishes processed with alternating stripes of ectodomain of fibronectin and leucine-rich transmembrane protein-2 (FLRT2) and control immunoglobulin G (IgG) fragment crystallizable region (Fc) protein. Both axons and cell bodies were strongly repelled from the FLRT2-coated stripe regions after 24 h of culture. Immunostaining with tau1 showed that ~90% of the neurons were distributed on the Fc-coated stripes compared to the FLRT2-Fc-coated stripes (~10%). This result indicates that FLRT2 has a strong repulsive effect on these neurons. This powerful method is applicable not only for primary cultured neurons but also for a variety of other cells, such as neuroblasts.

Dissociated hippocampal neurons from E15.5 mice were plated and cultured for 24 h on stripes of fluorescently labeled control Fc (Figure 3A-C) or FLRT2-Fc (Figure 3D-F) alternating with non-labeled control Fc. In both cases, the neurons were aggregated and extended their axons as bundles. On the control Fc/Fc stripes, the neurons were distributed evenly on the fluorescently labeled and non-labeled stripes, and they extended their axons in random direction...

Watch this Scientific Journal Video about Stripe Assay to Study the Attractive or Repulsive Activity of a Protein Substrate Using Dissociated Hippocampal Neurons at JoVE.com

Stripe Assay to Study the Attractive or Repulsive Activity of a Protein Substrate Using Dissociated Hippocampal Neurons

Axon guidance molecules regulate neuronal migration and targeted growth-cone navigation. We present a powerful method, the stripe assay, to assess the ability of guidance molecules to attract or repulse neurons. In this protocol, we demonstrate the stripe assay by showing FLRT2's ability to repel cultured hippocampal neurons.

Growing axons develop a highly motile structure at their tip, termed the growth cone. The growth cone contacts extracellular environmental cues to ...

The overall goal of the stripe assay is to asses the ability of specific proteins, such as guidance molecules, to attract or repel neurons. This method can help answer key questions in the field of axon guidance, such as, how neurons can migrate or extend their axons in response to the external cellular environment. The main advantage of this technique is that a large number of neurons can be visualized in a single experiment and many growth cones can be analyzed simultaneously.In preparation, boil four to eight silicone matrices in water for five minutes. Allow them to completely air dry under a laminar flow hood with their striped sides up. This takes about an hour.Continue working under the hood. Use compressed air or sticky tape to remove any dust on the striped side of the matrices. Then carefully place each matrix into a separate six centimeter plastic dish, striped side down.With one finger, press each matrix firmly. Avoid trapping air bubbles between the matrix and the dish. If the matrix fails to attach, repeat the process.Once attached, mark the location of the stripes on the bottom side of the culture dish. Prepare the fluorescently labeled recombinant proteins in PBS. Make 25 microliters for each striped dish.Let the mixture incubate at room temperature for 30 minutes. Next, for each dish, load a 22 gauge syringe with 25 microliters of diluted protein and inject it through the small hole on the side of the matrix. Avoid the injection of air bubbles into the matrices as they will disturb the binding of your proteins onto their dishes.Now, incubate the dishes for 30 minutes at 37 degrees Celsius in a cell culture incubator. After the incubation, deposit 300 microliters of PBS into the top slit of each matrix. Then aspirate the PBS from a small hole located on the side of each matrix to remove all the unattached recombinant proteins.Do not fully aspirate the PBS because the proteins will dry out. Perform this PBS rinse a total of three times. Next, carefully remove the matrix from each dish.Then immediately add about 100 microliters of controlled protein over the entire striped area, creating an alternate coating on each dish. Next, incubate the dishes for 30 minutes at 37 degrees Celsius. Meanwhile, on ice, thaw laminin and PBS at 20 micrograms per milliliter.After 30 minutes, wash the dishes three times with PBS. Then coat each dish with about 100 microliters of cold laminin and return the dishes to the incubator for another hour. An hour later, use PBS to wash the dishes three times again.After the third wash, add about 150 microliters of culture medium to each dish. Then incubate the dishes in a 5%carbon dioxide incubator at 37 degrees Celsius until they are next needed. After euthanizing a donor E15.5 mouse, cut the skin and skull sagittally at the mid-line of the head with surgical scissors and scoop the brain using a micro spatula.Carefully transfer the brain to a 60 milliliter petri dish containing three milliliters of HBSS. In the dish, use a scalpel to cut out the hemispheres including the cortex, hippocampus and striatum. Then dissect out the hippocampal regions by carefully removing the meninges.Transfer the dissected hippocampal tissues to a 15 milliliter centrifuge tube containing two milliliters of HBSS solution on ice. Six hippocampi from three embryos are sufficient for culturing neurons on eight dishes. With all of the hippocampus tissues collected, aspirate out the HBSS and replace it with two milliliters of trypsin-EDTA.Then incubate the tube for 15 minutes in a 37 degree Celsius water bath. Next, neutralize the trypsin activity by adding 500 microliters of fetal bovine serum. Then centrifuge the mixture at 100 Gs for five minutes.Remove the supernatant and wash the pellet with two milliliters of culture medium. Then centrifuge the mixture again and replace the medium with more medium. Now dissociate the tissue with gentle trituration using a one milliliter pipe head with a cut tip.Flow the tissues up and down through the pipe head tip about 10 times. Then use an uncut one milliliter tip and repeat the trituration to obtain a single cell suspension. Now isolate the single cells by pressing the suspension through an 80 to 100 micron mesh cell strainer.Then centrifuge the filtered suspension at 150 Gs for five minutes, aspirate out the supernatant and re-suspend the pellet in two milliliters of culture media. Count the cells using a hemocytometer and plate 10, 000 neurons in 150 microliters of culture media on each prepared stripe plate. Carefully deposit the suspension to cover the entire striped region.Dissociated hippocampal neurons from E15.5 mice were plated and cultured for 24 hours on stripes of flourescently labeled control protein FC, or the neurons were plated on stripes of FLRT2 FC alternating with non-labeled control FC.In both cases, the neurons were aggregated and extended their axons as bundles. On the FC control the neurons were distributed evenly and extended their axons in random directions. In contrast, when cultured with FLRT2 FC stripes, the axons avoided growing on the FLRT2 FC regions.Thus the extending axons grew mainly on the FC stripes. Higher magnification shows that the axons grew along the border with FLRT2 FC stripes, but did not extend into the FLRT2 territory. After watching this video you should have a good understanding of how to make striped carpet of the recommended proteins and asses the attractive or paras activity using the dissociated hippocampal neurons.Once mastered, this technique can be done in five hours if it is performed properly.

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JoVE Journal

Neuroscience

10.5K Görüntüleme.  Hamamatsu University School of Medicine. The overall goal of the stripe assay is to asses the ability of specific proteins, such as guidance molecules, to attract or repel neurons. This method can help answer key questions in the field of axon guidance, such as, how neurons can migrate or extend their axons in response to the external cellular environment. The main advantage of this technique is that a large number of neurons can be visualized in a single experiment and many growth cones can be analyzed simultaneously.In prep...