Name: stripe assay to study the attractive or repulsive activity of a protein substrate using dissociated hippocampal neurons
Uploaded: 2016-06-19T14:00:00
Description: 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

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>

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 ...

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