Name: an engulfment assay: a protocol to assess interactions between cns phagocytes and neurons
Uploaded: 2014-06-08T23:00:00
Description: Watch this Scientific Journal Video about An Engulfment Assay: A Protocol to Assess Interactions Between CNS Phagocytes and Neurons at JoVE.com

Arbeidet ble støttet med tilskudd fra Smith Family Foundation (BS), Dana Foundation (BS), John Merck Scholars Program (BS), ninds (RO1-NS-07100801, BS), NRSA (F32-NS-066698, DPS), Nancy Lurie Marks Foundation (DPS), NIH (P30-HD-18655; MRDDRC Imaging Core).

En. Antero Merking av RGC presynaptiske Innganger Merk: Alle forsøk med bruk av dyr ble anmeldt og overvåket av den institusjonelle dyr omsorg og bruk komité (IACUC) i samsvar med alle NIH retningslinjer. <ol><li> Steril feltet og instrumenter. </li><li> Anesthetize mus med 4 vol% isofluran i en pleksiglass induksjon kammer (dette vol% isofluran fungerer for neonatal-voksen mus). Observer mus nøye for å unngå over-bedøvelse. FORSIKTIG: Unngå innånding med et vakuum avfall gassevak...

<ol>
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For å måle fagocytose, må omsluttet materiale være merket på en slik måte at forskeren kan visualisere det en gang lysosomal degradering har oppstått. I tillegg er høy oppløsning kreves, etterfulgt av bruk av programvare som vil gjøre det mulig for forskeren til å visualisere volumet av hele cellen og kvantifisere innholdet. I denne protokollen, beskriver vi en svært pålitelig og kvantitativ metode for å måle phagocyte-mediert engulfment hjelp CTB konjugert til Alexa fargestoffer til å merke oppslukt mat...

<html> Synaptiske kretser remodel gjennom hele livet av et dyr. I utviklingen av hjernen, synapser dannes i overkant og må gjennomgå synaptiske beskjæring som innebærer selektiv fjerning av en undergruppe av synapser og opprettholdelse og forsterkning av disse synapser som forblir 8-10. Denne prosess er nødvendig for å oppnå den nøyaktige tilkoblings karakteristisk for den voksne nervesystemet. I den voksne, kan synapser også være plast, særlig i sammenheng med læring og hukommelse. De strukturelle korrelater med denne plastisitet er tenkt å omfatte tilsetning og / eller fjerning av dendrittutløperne og presynaptiske boutons 11-13. I tillegg til disse roller i sunt nervesystem, er synaptiske ombygging også involvert i nervesystem sykdommer / skader 12,14,15. For eksempel, etter ryggmargsskade, avkuttede axons må deretter renovere og danne nye synapser å oppnå funksjonelle utvinning 16-19. nt &quot;&gt; Emerging som en viktig del av synaptisk plastisitet er prosessen med fagocytose eller engulfment av synapser bestemt for fjerning 3,5,20. Vi har nylig viste dette fenomenet i sammenheng med synaptiske beskjæring i sunn, postnatal mus hjernen 7. Spesielt , microglia, bosatt CNS immunceller og fagocytter, ble vist å sluke presynaptiske inngangene under en topp periode og i et område av utviklings synaptiske beskjæring, barsel dorsal lateral geniculate nucleus (dLGN) i thalamus. Genetisk eller farmakologisk blokade av denne engulfment resulterte i vedvarende underskudd i synaptiske tilkobling. I denne protokollen, beskriver vi en pålitelig og svært kvantitativ analyse for å måle phagocyte-mediert engulfment av presynaptiske innganger. Ved anvendelsen av denne artikkelen, vil denne analysen bli presentert i sammenheng med utviklings retinogeniculate system, som inkluderer retinal ganglion celler (RGCs) bosatt i netthinnen somprojisere presynaptiske innganger til dLGN (figur 1A). Til å begynne med vil en lysosomal degradering bestandig antero merking strategi beskrives, som brukes til å visualisere RGC-spesifikke presynaptiske innganger i dLGN (figur 1) 7,21. Etter denne beskrivelse, en detaljert metode for avbildning og kvantitativt måle engulfment ved hjelp av konfokal mikroskopi i kombinasjon med 3-dimensjonal (3D) overflatevolumgjengivelse vil bli gitt. Denne metode er basert på fast vevspreparat, men kan også tilpasses for anvendelse i levende bildediagnostikk. Viktigere, mens analysen er blitt validert i sammenheng med den friske, postnatal retinogeniculate system, kan man anvende de samme teknikkene for å vurdere andre fagocytt-neuron interaksjoner i hele hjernen og i løpet av sykdommen, så vel som phagocyte funksjon i andre organsystemer.

<table border="0" cellpadding="0" cellspacing="0" width="533">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:144px;">Heat pad</td>
			<td style="width:159px;">Vet Equip, Inc.</td>
			<td style="width:168px;">965500&#160;</td>
			<td style="width:63px;"></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:144px;">Warm water source for heat pad</td>
			<td style="width:159px;">Kent Scientific</td>
			<td style="width:168px;">TP-700</td>
			<td></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:144px;">Stereo microscope</td>
			<td style="width:159px;">DSC Optical</td>
			<td style="width:168px;">Zeiss Opmi -6 Surgical Microscope</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="40" rowspan="2" style="height:40px;width:144px;">Sliding microtome with freezing stage</td>
			<td rowspan="2" style="width:159px;">Leica</td>
			<td rowspan="2" style="width:168px;">SM2010 R</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:144px;">Microtome blade</td>
			<td style="width:159px;">Leica</td>
			<td style="width:168px;">14021607100</td>
			<td></td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:144px;">Fluorescent dissecting microscope</td>
			<td style="width:159px;">Nikon</td>
			<td style="width:168px;">SMZ800 with Epi-fluorescence attachment</td>
			<td></td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:144px;">Spinning disk confocal microscope</td>
			<td style="width:159px;">Perkin Elmer</td>
			<td style="width:168px;">UltraView Vox Spinning Disk Confocal</td>
			<td></td>
		</tr>
		<tr height="51">
			<td height="51" style="height:51px;width:131px;">10 &#181;l Hamilton gas tight syringes</td>
			<td style="width:131px;">Hamilton</td>
			<td style="width:116px;">80030</td>
			<td style="width:144px;">Use a different syringe for each color dye/tracer</td>
		</tr>
		<tr height="51">
			<td height="51" style="height:51px;width:131px;">Hamilton needles</td>
			<td style="width:131px;">Hamilton</td>
			<td style="width:116px;">7803-05, specifications: blunt, 1.5&#34;</td>
			<td style="width:144px;"></td>
		</tr>
		<tr height="20">
			<td height="54" rowspan="3" style="height:54px;width:131px;">Alexa-conjugated cholera toxin &#946; subunit (CTB)</td>
			<td rowspan="3" style="width:131px;">Invitrogen</td>
			<td style="width:116px;">488: C22841</td>
			<td rowspan="3" style="width:144px;">Reconstitute in sterile saline, 80 &#181;l (488), 100 &#181;l (594), 20 &#181;l (647)</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:116px;">594: C22842</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:116px;">647: C34778</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:131px;">Phosphate Buffered Saline&#160;(PBS)</td>
			<td style="width:131px;">Sigma</td>
			<td>P4417-50TAB</td>
			<td style="width:144px;"></td>
		</tr>
		<tr height="73">
			<td height="73" style="height:73px;width:131px;">Neomycin and Polymyxin B Sulfates and Bacitracin Zinc Ophthalmic Ointment USP (antibiotic ointment)</td>
			<td style="width:131px;">Bausch &#38; Lomb</td>
			<td style="width:116px;">24208-780-55</td>
			<td style="width:144px;"></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:131px;">30.5 gauge needle</td>
			<td style="width:131px;">Becton Dickinson</td>
			<td style="width:116px;">305106</td>
			<td rowspan="3" style="width:144px;"></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:131px;">Spring scissors</td>
			<td style="width:131px;">Roboz</td>
			<td style="width:116px;">RS-5630</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:131px;">Cotton-tipped applicator</td>
			<td style="width:131px;">Fisher</td>
			<td style="width:116px;">23-400-125</td>
		</tr>
		<tr height="21">
			<td height="104" rowspan="2" style="height:104px;width:131px;">Paraformaldeyde (PFA)</td>
			<td rowspan="2" style="width:131px;">Electron Microscopy Sciences</td>
			<td rowspan="2" style="width:116px;">15710</td>
			<td rowspan="2" style="width:144px;">Dilute 16%to 4% in PBS. Paraformaldehye is toxic, use&#160; in a fume hood and wear personal protective equipment.</td>
		</tr>
		<tr height="83">
		</tr>
		<tr height="36">
			<td height="138" rowspan="4" style="height:138px;width:131px;">Dissection tools &#8211; scissors, forceps, spatula</td>
			<td style="width:131px;">Small scissors: Fine Science Tools</td>
			<td style="width:116px;">Small scissors:14370-22</td>
			<td rowspan="4" style="width:144px;"></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:131px;">Large scissors: Roboz</td>
			<td style="width:116px;">Large scissors: RS-6820</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:131px;">#55 forceps: Fine Science Tools</td>
			<td style="width:116px;">#55 forceps: 11255-20</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:131px;">Spatula: Ted Pella, Inc.</td>
			<td style="width:116px;">Spatula: 13504</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:131px;">Sucrose</td>
			<td style="width:131px;">Sigma</td>
			<td style="width:116px;">S8501-5KG</td>
			<td style="width:144px;">Make 30% sucrose in PBS (weight/vol)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:131px;">OCT Compound</td>
			<td style="width:131px;">VWR</td>
			<td style="width:116px;">25608-930</td>
			<td style="width:144px;"></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:131px;">Weigh boat</td>
			<td style="width:131px;">USA Scientific</td>
			<td style="width:116px;">2347-1426</td>
			<td style="width:144px;"></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:131px;">24-well plates</td>
			<td style="width:131px;">BD Biosciences</td>
			<td style="width:116px;">353047</td>
			<td style="width:144px;"></td>
		</tr>
		<tr height="87">
			<td height="87" style="height:87px;width:131px;">Sodium phosphate monobasic</td>
			<td style="width:131px;">Sigma</td>
			<td style="width:116px;">S6566-500G</td>
			<td rowspan="2" style="width:144px;">Make 0.2 M sodium phosphate monobasic (PB-A) in ddH20 and 0.2 M sodium phosphate dibasic (PB-B) in ddH20.&#160; To make 0.1 M PB, combine 19 ml PB-A and 81 ml PB-B, fill to 200 ml with ddH20&#160;&#160;</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:131px;">Sodium phosphate dibasic&#160;</td>
			<td style="width:131px;">Sigma</td>
			<td>S5136-500G</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:131px;">Coverslips, 22 X 50 mm, No. 1.5</td>
			<td style="width:131px;">VWR</td>
			<td style="width:116px;">48393 194</td>
			<td style="width:144px;"></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:131px;">Charged microscope slide</td>
			<td style="width:131px;">VWR</td>
			<td style="width:116px;">48311-703</td>
			<td style="width:144px;"></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:131px;">Vectasheild</td>
			<td style="width:131px;">Vector Laboratories</td>
			<td style="width:116px;">H-1200</td>
			<td style="width:144px;"></td>
		</tr>
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an engulfment assay: a protocol to assess interactions between cns phagocytes and neurons

Fagocytose er en prosess hvor en celle omslutter materialet (hel celle, deler av en celle, rusk etc.) i sin omliggende ekstracellulære miljø og deretter fordøyer dette materialet, vanligvis gjennom lysosomal degradering. Mikroglia er beboeren immunceller i sentralnervesystemet (CNS) som fagocytisk funksjon er blitt beskrevet i en lang rekke forhold fra nevrodegenerative sykdommer (for eksempel beta-amyloid klaring i Alzheimers sykdom) til utvikling av det friske hjernen (f.eks synaptiske beskjæring) 1-6. Følgende protokoll er en engulfment analyse utviklet for å visualisere og kvantifisere microglia-mediert engulfment av presynaptiske innganger i den tredje muse retinogeniculate system 7. Selv om denne assay ble brukt for å vurdere microglia funksjon i denne spesielle sammenheng, kan en lignende tilnærming brukes til å vurdere andre fagocytter i hele hjernen (f.eks astrocytter) og resten av legemet(f.eks perifere makrofager) samt andre sammenhenger der oppstår synaptic ombygging (f.eks hjerneskade / sykdom).

Nylig har vi brukt denne engulfment analysen til å visualisere og kvantifisere microglia-mediert engulfment av presynaptiske innganger i utviklings retinogeniculate systemet (figur 1) 7. RGCs fra CX3CR1-EGFP heterozygote mus ble anterogradely spores med CTB-594 og CTB-647 til venstre og høyre øyne, henholdsvis. Etter denne tracing, ble EGFP-positive microglia innenfor dLGN avbildes. Disse bildene ble deretter overflaten-rendret for volummålinger. Ved hjelp av ...

Watch this Scientific Journal Video about An Engulfment Assay: A Protocol to Assess Interactions Between CNS Phagocytes and Neurons at JoVE.com

An Engulfment Assay: A Protocol to Assess Interactions Between CNS Phagocytes and Neurons

Mikroglia er beboeren immunceller i sentralnervesystemet (CNS) med en høy kapasitet til å phagocytose eller sluker materiale i sitt ekstracellulære miljø. Her blir en bredt anvendbar, pålitelig og meget kvantitativ analyse for å visualisere og måle microglia-mediert engulfment av synaptiske komponenter beskrevet.

En engulfment analysen: En protokoll for å vurdere samspillet mellom CNS Fagocytter og Neuroner

Phagocytosis is a process in which a cell engulfs material (entire cell, parts of a cell, debris, etc.) in its surrounding extracellular environment and ...

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