Stripe Assay til at studere den attraktiv eller Repulsive aktivitet af et protein underlaget med Dissocierede hippocampusneuroner
Summary
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.
Abstract
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.
Introduction
Axon vejledning er den proces, hvorved nydannede neuroner sender axoner til deres target under udviklingen af nervesystemet 1,2. Udviklingslande axoner bære en meget motile struktur på deres spids kaldes kegle vækst. Keglen vækst registrerer ekstracellulære stikord til at navigere Axon vej. Guidance molekyler, såsom slids, semaphorin og ephriner, kan tiltrække eller frastøde axoner afhængigt af deres interaktion med egnede receptorer og co-receptorer på Axon 1,3,4. De aktiverede receptorer overfører signaler til væksten kegle, der påvirker dets cytoskeletorganisation for Axon og vækst-kegle bevægelser.
Der er udviklet forskellige metoder til at vurdere virkningen af tiltrækkende og frastødende molekyler. Kemo-tiltrækkende og afskrækningsmidler kan administreres i vækst / dyrkningsmediet med en gradient koncentration (f.eks., Dunns kammer eller u–slides) 5,6, i en meget koncentreret stedet ved mikro-pipette (f.eks., drejning assay) 7 eller ved en homogen koncentration af bad program (f.eks., vækst kegle sammenbrud assay) 8,9.
Andre metoder omfatter en stribe assay eller mikrokontakt-printing (μCP), hvori en kemo-tiltrækningsstof eller frastødende coates på overfladen af en plade som substrat 10-12. Thestripe assay blev oprindeligt udviklet af Bonhoeffer og kolleger i 1987 for at analysere topografisk kortlægning i chick retino-tectal systemet 13. Den oprindelige metode krævede et vakuumsystem til kappeproteiner på polycarbonat Nucleopore membraner ved anvendelse stribede og i indgreb matricer. I senere versioner, blev de rekombinante proteiner er direkte trykt på overfladen af en agarplade i et stribet mønster under anvendelse smal spalte silicium matricer 14,15. For nylig har forskellige forskergrupper succes anvendt denne stribe assay til analysen af axon vejledning molekyle aktiviteter 16-21.
<p class = "jove_content"> Her præsenterer vi den detaljerede protokol for en stribe forsøg, der måler den tiltrækning eller frastødning af axon vejledning molekyler til dissocierede hippocampus neuroner. Især kan denne metode anvendes i minimalt udstyrede indstillinger laboratorium. Til dette assay alternerende striber af en fluorescens-mærket substrat og et kontrolprotein genereres på en plastskål anvendelse af en silicium matrix med 90 um slidser og belagt med laminin. I vores demonstration, dissocieret hippocampale neuroner fra E15.5 mus blev dyrket på alternerende striber af rekombinant ektodomæne af fibronectin og leucin-rige transmembrane protein-2 (FLRT2) og styre Fc-protein 21. Efter 24 timers dyrkning blev både axoner og celle organer neuronerne stærkt frastødt fra FLRT2 striber. Farvning med et anti-Tau1 antistof viste, at ~ 90% af neuronerne blev fordelt på de Fc-belagte områder, sammenlignet med ~ 10% på FLRT2-Fc, hvilket viser, at FLRT2 har en stærk frastødendefunktion for hippocampale neuroner 21.Protocol
Representative Results
Discussion
Denne protokol beskriver en stribe assay, der anvender rekombinant protein og dissocierede neuroner fra E15.5 musehippocampus. Dette assay muliggør effektiv observation af frastødende, attraktive, eller neutrale reaktioner af neuroner til et rekombinant protein af interesse anbringes i et stribet mønster. En stor fordel ved denne protokol er den enkle metode til generering striberne, hvor proteinet er direkte trykt på overfladen af en plastik skål, sammenlignet med den traditionelle metode, som kræver særlige mat…
Divulgations
The authors have nothing to disclose.
Acknowledgements
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.).
Materials
| 15 mL centrifuge tube | Violamo | 1-3500-01 | |
| 4% Paraformaldehyde (PFA) | Nacalai | 01954-85 | |
| Alexa Fluor 488 Goat anti-human IgG antibody | Thermo Scientific | A11013 | |
| Alexa Fluor 594 Donkey anti-mouse IgG antibody | Thermo Scientific | A-21203 | Dilution 1/500 |
| Anti-Tau1 antibody | Chemicon | MAB3420 | Dilution 1/200 |
| Antifade | Thermo Scientific | P7481 | Alternative mounting media may be used |
| B27 supplement | Thermo Scientific | 17504-044 | Dilution 1/50 |
| Bovine serum albumin | Sigma | 01-2030-2 | |
| Cell strainer 100 um | BD Falcon | 352360 | |
| Centrifugation machine | Kubota | 2410 | |
| Cover glass 18mmx18mm | Matsunami | 18×18 mm No. 1 | |
| DAKO pen | DAKO | S2002 | Alternative water-repellent pen may be used |
| Disposable scalpel | Feather | 2975#11 | |
| FBS | Thermo Scientific | 10437-028 | |
| Fluorecent microscope | Nikon | E600 | |
| Forceps No. 5 | Fine Science Tools | 11254-20 | |
| GlutaMAX | Thermo Scientific | 35050-061 | Dilution 1/200 |
| Hamilton Syringe | Hamilton | 805N | 22 gauge, 50 uL |
| HBSS | Thermo Scientific | 14170-112 | |
| Human IgG, Fc Fragment | Jackson | 009-000-008 | |
| Laminin | Thermo Scientific | 23017-015 | |
| Neurobasal | Thermo Scientific | 21103-049 | |
| Normal Donkey Serum | Jackson | 017-000-121 | |
| PBS | Nacalai | 14249-24 | |
| Penicillin-Streptomycin | Thermo Scientific | 15070-063 | Dilution 1/100 |
| Plastic culture dish, 60 mm | Thermo Scientific | 150288 | |
| Silicone Matrices | Available and purchasable from Prof. Martin Bastmeyer (bastmeyer@kit.edu) | ||
| Stereo Microscope | Olympus | SZ61 | |
| Tip, 1000 uL | Watson | 125-1000S | |
| Transparent sticky tape | Tesa | 57315 | Alternative sticky tape may be used |
| Triton X-100 | Sigma | T8787 | |
| Trypan blue, 0.4% | Bio-Rad | 145-0013 | |
| Trypsin/EDTA | Thermo Scientific | 25300-054 | |
| Culture medium | Neurobasal supplemented with B27, GlutaMAX and Penicillin-Streptomycin. |
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