Name: a galvanotaxis assay for analysis of neural precursor cell migration kinetics in an externally applied direct current electric field
Uploaded: 2012-10-13T14:00:00
Description: Watch this Scientific Journal Video about A Galvanotaxis Assay for Analysis of Neural Precursor Cell Migration Kinetics in an Externally Applied Direct Current Electric Field at JoVE.com

Dette arbejde er finansieret af Natural Sciences and Engineering Research Council of Canada (tilskud # 249.669, og # 482.986) og Heart and Stroke Foundation of Canada (tilskud # 485.508). Forfatterne takker Youssef El-Hayek og Dr. Qi Wan for deres assistance i udviklingen af ​​forsøgsprotokoller.

Alle procedurer, der involverer håndtering af dyr blev godkendt af University of Toronto Animal Care udvalg i overensstemmelse med de institutionelle retningslinjer (protokol nr. 20.009.387). Følgende metoder bør udføres med sterile redskaber og teknikker, i en laminar strømningshætte givet fald. I protokollen teksten nedenfor, henviser udtrykket &quot;EFH-SFM&quot; til serumfrit medium suppleret med epidermal vækstfaktor, basisk fibroblastvækstfaktor og heparin. EFH-SFM bruges, når...

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Denne protokol er blevet tilpasset fra de veletablerede metoder til tidligere undersøgelser 7-9. Galvanotactic kamre kan konstrueres ved anvendelse af en række forskellige teknikker, herunder konstruktionen af et separat glas godt for indespærring af cellepodning, eller ved hjælp af CO 2-laser ablation til mikrofabrikation af den centrale trug 10,11. Nogle teknikker kan være mere arbejdskrævende eller dyre end andre. Vi har beskrevet en enkel og omkostningseffektiv analyse for at k...

<table border="1"><tbody><tr><td height="44"> Genstandens navn </td><td> Firma </td><td> Katalognummer </td><td> Kommentarer </td></tr><tr><td></td><td></td><td></td><td> Neural precursor celleisolering </td></tr><tr><td> 2 M NaCI </td><td> Sigma </td><td> S5886 </td><td> 11,688 g opløses i 100 ml dH2O </td></tr><tr><td> 1M KCl </td><td> Sigma </td><td> P5405 </td><td> 7,456 g opløst i 100 ml dH2O </td></tr><tr><td> 1 M MgCl2 </td><td> Sigma </td><td> M2393 </td><td> 20,33 g opløses i 100 ml dH2O </td></tr><tr><td> 155 mM NaHCO3 </td><td> Sigma </td><td> S5761 </td><td> 1,302 g opløst i 100 ml dH2O </td></tr><tr><td> 0.5M Glucose </td><td> Sigma </td><td> G6152 </td><td> 9,01 g opløst i 100ml dH2O </td></tr><tr><td> 108 mM CaCl2 </td><td> Sigma </td><td> C7902 </td><td> 1,59 g opløst i 100 ml dH2O </td></tr><tr><td> Penicillin-streptomycin </td><td> Gibco </td><td> 15.070 </td><td></td></tr><tr><td> Bovin pancreas trypsin </td><td> Sigma </td><td> T1005 </td><td></td></tr><tr><td> Får testikler hyaluronidase </td><td> Sigma </td><td> H6254 </td><td></td></tr><tr><td> Kynurensyre </td><td> Sigma </td><td> K3375 </td><td></td></tr><tr><td> Ovimucoid trypsininhibitor </td><td> Worthington </td><td> LS003086 </td><td></td></tr><tr><td> DMEM </td><td> Invitrogen </td><td> 12100046 </td><td></td></tr><tr><td> F12 </td><td> Invitrogen </td><td> 21700075 </td><td></td></tr><tr><td> 30% Glucose </td><td> Sigma </td><td> G6152 </td><td></td></tr><tr><td> 7,5% NaHCO3 </td><td> Sigma </td><td> S5761 </td><td></td></tr><tr><td> 1M HEPES </td><td> Sigma </td><td> H3375 </td><td> 23,83 g opløses i 100 ml dH2O </td></tr><tr><td> L-glutamin </td><td> Gibco </td><td> 25.030 </td><td></td></tr><tr><td> EGF </td><td> Invitrogen </td><td> PMG8041 </td><td> Opløses i 1 ml hormon-mix og prøve i 20 ul enheder. </td></tr><tr><td> FGF </td><td> Invitrogen </td><td> PHG0226 </td><td> Rekonstitueres i 0,5 ml hormon-mix-og alikvot i 20 ul enheder. </td></tr><tr><td> Heparin </td><td> Sigma </td><td> H3149 </td><td></td></tr><tr><td> Apo-transferrin </td><td> R &amp; D Systems </td><td> 3188-AT </td><td> 0,1 g opløst i 4 ml dH 2 0 </td></tr><tr><td> Putrescin </td><td> Sigma </td><td> P7505 </td><td> Opløs 9,61 mg til Apo-transferrin sålution </td></tr><tr><td> Insulin </td><td> Sigma </td><td> I5500 </td><td> Opløs 25 mg i 0,5 ml 0,1 N HCI og tilsættes til 3,5 ml dH 2 0 </td></tr><tr><td> Selen </td><td> Sigma </td><td> S9133 </td><td></td></tr><tr><td> Progesteron </td><td> Sigma </td><td> P6149 </td><td></td></tr><tr><td> Standard Dissektionsværktøj </td><td> Fin Science Tools </td><td></td><td></td></tr><tr><td> Dissection mikroskop </td><td> Zeiss </td><td> STEMI 2000 </td><td></td></tr><tr><td></td><td></td><td></td><td> Galvanotaxis Chamber Forberedelse </td></tr><tr><td> Square glasafskærmning dias </td><td> VWR </td><td> 16.004 </td><td></td></tr><tr><td> 6 N saltsyre </td><td> VWR </td><td> BDH3204-1 </td><td></td></tr><tr><td> Høj vakuum fedt </td><td> Dow Corning </td><td></td><td></td></tr><tr><td> 60 mm petriskåle </td><td> Fisher Scientific </td><td> 0875713A </td><td></td></tr><tr><td> Poly-L-lysin </td><td> Sigma </td><td> P4707 </td><td></td></tr><tr><td> Matrigel </td><td> BD Biosciences </td><td> 354.234 </td><td> Thaw og alikvot i 150 ul enheder </td></tr><tr><td> FBS </td><td> Invitrogen </td><td> 10082139 </td><td> Brug kun hvis inducere NPC differentiering, ellers bruge SFM + EFH dyrkningsmedier som anført ovenfor </td></tr><tr><td> Optælling mikroskop </td><td> Olympus </td><td> CKX41 </td><td></td></tr><tr><td></td><td></td><td></td><td> Levende Cell Time-Lapse Imaging </td></tr><tr><td> Sølvtråd </td><td> Alfa Aesar </td><td> 11.434 </td><td></td></tr><tr><td> UltraPure Agarose </td><td> Invitrogen </td><td> 15510-027 </td><td></td></tr><tr><td> Heat Inactivated FBS </td><td> Sigma </td><td> 16140071 </td><td></td></tr><tr><td> PVC </td><td> Fisher Scientific </td><td> 80000006 </td><td> 3/32 &quot;diameter x 5/32&quot; OD </td></tr><tr><td> Blegemiddel </td><td> Clorox </td><td></td><td></td></tr><tr><td> 10 cc sprøjte </td><td> BD </td><td> 309.604 </td><td></td></tr><tr><td> 18 gauge nål </td><td> BD </td><td> 305.195 </td><td></td></tr><tr><td> Dremel boremaskine </td><td> Dremel </td><td> Model 750 </td><td></td></tr><tr><td> Inverteret mikroskop udstyret med befugtet, inkuberet kammer </td><td> Zeiss </td><td> Axiovert-200M </td><td></td></tr><tr><td></td><td></td><td></td><td> Opskrifter <table border="1"><tbody><tr><td> Item </td><td> Volumen </td></tr><tr><td> 2 M NaCI </td><td> 6,2 ml </td></tr><tr><td>1M KCl </td><td> 0,5 ml </td></tr><tr><td> 1 M MgCl2 </td><td> 0,32 ml </td></tr><tr><td> 155MM NaHCO3 </td><td> 16,9 ml </td></tr><tr><td> 1M Glucose </td><td> 1 ml </td></tr><tr><td> 108 mM CaCl2 </td><td> 0,09256 ml </td></tr><tr><td> Penicillin-streptomycin </td><td> 1 ml </td></tr><tr><td> Autoklaveret vand </td><td> 74 ml </td></tr></tbody></table> Kunstig cerebrospinalvæske <table border="1"><tbody><tr><td> Item </td><td> Volumen eller Masse </td></tr><tr><td> Kunstig cerebrospinalvæske </td><td> 30 ml </td></tr><tr><td> Bovin pancreas trypsin </td><td> 40 mg </td></tr><tr><td> Får testikler hyaluronidase </td><td> 22,8 mg </td></tr><tr><td> Kynurensyre </td><td> 5 mg </td></tr></tbody></table> &lt;strong&gt; trypsinopløsning <table border="1"><tbody><tr><td> Item </td><td> Volumen eller Masse </td></tr><tr><td> SFM </td><td> 15 ml </td></tr><tr><td> Ovimucoid trypsininhibitor </td><td> 10 mg </td></tr></tbody></table> Trypsininhibitor Solution <table border="1"><tbody><tr><td> Item </td><td> Volumen </td></tr><tr><td> Autoklaveret vand </td><td> 37 ml </td></tr><tr><td> 10X DMEM/F12 </td><td> 10 ml </td></tr><tr><td> 30% Glucose </td><td> 2 ml </td></tr><tr><td> 7,5% NaHCO3 </td><td> 1,5 ml </td></tr><tr><td> 1M HEPES </td><td> 0,5 ml </td></tr><tr><td> Transferrin, Putrescin opløsning </td><td> 4 ml </td></tr><tr><td> 25 mg insulinopløsning </td><td> 4 ml </td></tr><tr><td> Selen </td><td> 100 &amp; mu; L </td></tr><tr><td> Progesteron </td><td> 100 pi </td></tr></tbody></table> Hormone Mix (100 ml i alt, opbevares ved -20 ° C) <table border="1"><tbody><tr><td> Item </td><td> Volumen </td></tr><tr><td> Autoklaveret vand </td><td> 37,5 ml </td></tr><tr><td> 10X DMEM/F12 (3:1) </td><td> 5 ml </td></tr><tr><td> 30% Glucose </td><td> 1 ml </td></tr><tr><td> 7,5% NaHCO3 </td><td> 0,75 ml </td></tr><tr><td> 1M HEPES </td><td> 0,25 ml </td></tr><tr><td> Hormon mix </td><td> 5 ml </td></tr><tr><td> L-glutamin </td><td> 0,5 ml </td></tr><tr><td> Penicillin-streptomycin </td><td> 0,5 ml </td></tr></tbody></table> Serum Free Media EFH-SFM: tilføj 10 gl af EGF, 10 pi FGF, og 3,66 pi Heparin FBS-SFM: Tilsæt 0,5 ml FBS <table border = &quot;1&quot;&gt; <tbody><tr><td> Item </td><td> Volumen </td></tr><tr><td> Matrigel </td><td> 150 ul </td></tr><tr><td> SFM </td><td> 3,6 ml </td></tr></tbody></table> Matrigel Solution Matrigel aliquot anbringes i en kasse med is og fik lov at optø langsomt i løbet af 4-5 timer til dannelse af en viskos væske før blanding med SFM. Dette vil sikre dannelsen af ​​et glat lag Matrigel substrat. Hvis ikke optøs langsomt, vil det resulterende substrat indeholde klumper af Matrigel, eventuelt hindre cellemigrering. <table border="1"><tbody><tr><td> Item </td><td> Volumen eller Masse </td></tr><tr><td> UltraPure Agarose </td><td> 300 mg i 10 ml ddH 2 0 </td></tr><tr><td> SFM Varmeinaktiveret FBS </td><td> 8 ml 2 ml </td></tr></tbody></table> MatrigelOpløsningen blandes 8 ml SFM med 2 ml varmeinaktiveret FBS i en 15 cc Falcon-rør. Mix agarose med 10 ml ddH 2 0 i en Erlenmeyerkolbe, og opvarmes i en mikrobølgeovn i 30 sek i 10 sek intervaller, som sikrer at fjerne opløsningen fra mikrobølge efter hver 10 sekunders interval og blandes grundigt. Efter den endelige 10 sekunders mikrobølge periode agaroseopløsning blandes med SFM / FBS opløsning og opbevares i 57 ° C vandbad. </td></tr></tbody></table>

a galvanotaxis assay for analysis of neural precursor cell migration kinetics in an externally applied direct current electric field

Opdagelsen af ​​neurale stam-og progenitorceller (samlet betegnet neurale precursorceller) (NPC) i den voksne pattedyrhjernen har ført til en omfattende forskning med henblik på at udnytte de multipotente og proliferative egenskaber af disse celler til udvikling af neuroregenerative strategier. Et afgørende skridt for en vellykket gennemførelse af sådanne strategier er mobilisering af NPCs mod en læsion site efter exogent transplantation eller at øge svar af de endogene prækursorer, der findes i periventricular region af CNS. Derfor er det vigtigt at forstå de mekanismer, der fremmer, vejlede og forbedre NPC migration. Vores arbejde fokuserer på udnyttelse af jævnstrøms elektriske felter (dcEFs) at fremme og direkte NPC migration - et fænomen kendt som galvanotaxis. Endogene fysiologiske elektriske felter fungerer som kritiske signaler for cellemigrering under normal udvikling og sårheling. Farmakologisk afbrydelse aftrans-neuralrøret potentiale i Axolotl embryoner forårsager alvorlige udviklingsmæssige misdannelser 1. I forbindelse med sårheling, er hastigheden for reparation af sårede hornhinde direkte korreleret med størrelsen af den epitel sår potentiale, der opstår efter beskadigelse, som det fremgår af farmakologisk forøgelse eller afbrydelse af denne dcEF 2-3. Vi har vist, at voksne subependymale NPCs undergår hurtig og rettet katodisk migration in vitro, når de udsættes for et eksternt påført dcEF. I denne protokol beskriver vi vores laboratorium har teknikker til at skabe en enkel og effektiv galvanotaxis assay for høj opløsning, langvarig observation af direkte cellelegemet translokation (migration) på en enkelt-celle niveau. Dette assay ville være egnet til at undersøge de mekanismer, der regulerer dcEF transduktion ind cellulære motilitet ved anvendelse af transgene eller knockout-mus, korte interfererende RNA eller specifikke receptor-agonister / antagonister.

Kinematisk analyse viser, at i nærværelse af et 250 mV / mm dcEF, udifferentierede NPCs udviser stærkt rettet og hurtig galvanotaxis mod katoden (figur 5A, Movie 1). I mangel af en dcEF, er tilfældige bevægelse af cellerne observeret (fig. 5B, Movie 2). På dette feltstyrke, migrerer&gt; 98% af udifferentierede NPCs for hele 6-8 timer, som de er afbildet, og da døde celler ikke migrerer dette tyder på, at de forbliver levedygtige i denne periode i fravær eller nærvær af en dcE...

Watch this Scientific Journal Video about A Galvanotaxis Assay for Analysis of Neural Precursor Cell Migration Kinetics in an Externally Applied Direct Current Electric Field at JoVE.com

A Galvanotaxis Assay for Analysis of Neural Precursor Cell Migration Kinetics in an Externally Applied Direct Current Electric Field

I denne protokol vi vise hvordan man opfører brugerdefinerede kamre, der tillader anvendelsen af ​​en jævnstrøm elektrisk felt for at aktivere time-lapse imaging af voksen hjerne afledt neural precursorcelletype translokation under galvanotaxis.

En Galvanotaxis Assay for Analyse af neurale Prækursormaterialer cellemigration Kinetics i en eksternt påført Direct Current Electric Field

The discovery of neural stem and progenitor  cells (collectively termed neural precursor cells) (NPCs) in the adult  mammalian brain has led to a body of ...

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Regeneration in the peripheral nervous system (PNS) is widely studied both for its relevance to human disease and to understand the robust regenerative ...

Analysis of Neural Crest Migration and Differentiation by Cross-species Transplantation

Analyse af Neural Crest migrering og differentiering af Cross-arter Transplantation

Avian embryos provide a unique platform for studying many vertebrate developmental processes, due to the easy access of the embryos within the egg. ...

The Neuroblast Assay: An Assay for the Generation and Enrichment of Neuronal Progenitor Cells from Differentiating Neural Stem Cell Progeny Using Flow Cytometry

Den neuroblast Assay: Et assay for generering og berigelse af neuronale progenitorceller fra Differentiering neural stamcelle Afkom anvendelse af flowcytometri

Neural stem cells (NSCs) can be isolated and expanded in large-scale, using the neurosphere assay and differentiated into the three major cell types of ...

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

Isolering og dyrkning af neurale kamceller fra embryonale Murin 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 ...

A Quantitative Cell Migration Assay for Murine Enteric Neural Progenitors

En kvantitativ Cell Migration Assay for Murine Enteriske Neural Progenitorer

Neural crest cells (NCC) are a transient and multipotent cell population that originates from the dorsal neural tube and migrates extensively throughout ...

Long-term Behavioral Tracking of Freely Swimming Weakly Electric Fish

Langsigtet Behavioral Tracking af Frit Svømning Svagt Electric Fish

Long-term behavioral tracking can capture and quantify natural animal behaviors, including those occurring infrequently. Behaviors such as exploration and ...

Remotely Supervised Transcranial Direct Current Stimulation: An Update on Safety and Tolerability

Fjernt overvåget transkranial direkte nuværende Stimulation: En opdatering på sikkerheden og tolerabiliteten

The remotely supervised tDCS (RS-tDCS) protocol enables participation from home through guided and monitored self-administration of tDCS treatment while ...