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

Arbejdet blev støttet af tilskud fra Smith Family Foundation (BS), Dana Foundation (BS), John Merck Scholars Program (BS), NINDS (RO1-NS-07.100.801, BS), NRSA (F32-NS-066.698, DPS) Nancy Lurie Marks Foundation (DPS), NIH (P30-HD-18655, MRDDRC Imaging Core).

1.. Anterograd Mærkning af RGC Præsynaptiske indgange Bemærk: Alle eksperimenter involverer brug af dyr blev gennemgået og overvåget af den institutionelle Dyrepleje og brug udvalg (IACUC) i overensstemmelse med alle NIH retningslinjer. <ol><li> Sterilisere felt og instrumenter. </li><li> Bedøver mus med 4 vol% isofluran i en plexiglas induktion kammer (dette vol% isofluran arbejder for neonatalt voksne mus). Overhold mus nøje for at undgå over-bedøve. ADVARSEL: Undgå indånding ...

<ol>
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For nøjagtigt at måle fagocytose skal opslugt materiale skal mærkes på en sådan måde, at forskeren kan visualisere det, når lysosomal nedbrydning har fundet sted. Desuden er høj opløsning nødvendig, efterfulgt af anvendelse af software, der vil gøre det muligt for forskeren at visualisere mængden af ​​hele cellen og kvantificere dets indhold. I denne protokol, beskriver vi en yderst pålidelig og kvantitativ metode til måling phagocyt-medieret engulfment hjælp CTB konjugeret til Alexa farvestoffer til ...

<html> Synaptiske kredsløb remodel gennem hele livet af et dyr. I hjernens udvikling, synapser dannes i overskud og skal gennemgå synaptisk beskæring, som indebærer selektiv fjernelse af en delmængde af synapser og opretholdelse og styrkelse af disse synapser der forbliver 8-10. Denne proces er nødvendig for at opnå den præcise tilslutningsmuligheder karakteristisk for voksne nervesystem. I den voksne kan synapser også være plast, især i forbindelse med indlæring og hukommelse. De strukturelle korrelater denne plasticitet menes at omfatte tilsætning og / eller fjernelse af dendritiske Torner og præsynaptiske boutons 11-13. Ud over disse roller i sundt nervesystem er synaptisk remodeling også involveret i nervesystem / tilskadekomst 12,14,15. For eksempel, efter rygmarvsskade, overskårne axoner skal derefter omforme og danne nye synapser at opnå funktionel genopretning 16-19. nt &quot;&gt; Emerging som et vigtigt aspekt af synaptisk plasticitet er processen med fagocytose eller engulfment af synapser bestemt til fjernelse 3,5,20. Vi har for nylig viste dette fænomen i forbindelse med synaptisk beskæring i den sunde, postnatal mus hjerne 7. Specifikt , mikroglia, de bosiddende CNS immunceller og fagocytter, viste sig at opsluge præsynaptiske input i løbet af en periode med spidsbelastning og i en region i udviklingsmæssig synaptisk beskæring, den postnatale dorsale laterale geniculate kerne (dLGN) i thalamus. genetisk eller farmakologisk blokade af denne engulfment resulterede i vedvarende underskud i synaptisk forbindelse. I denne protokol, beskriver vi en pålidelig og yderst kvantitativ analyse for at måle phagocyt-medieret engulfment af præsynaptiske indgange. Ved anvendelsen af ​​denne artikel, vil denne analyse blive præsenteret i forbindelse med den udvikling retinogeniculate system, som omfatter retina-ganglieceller (rGCS) med bopæl i nethinden,projicere præsynaptiske input til dLGN (figur 1A). Til at begynde, vil en lysosomal nedbrydning-resistent anterograd mærkning strategi beskrives, hvilket bruges til at visualisere RGC-specifikke præsynaptiske inputs i dLGN (figur 1) 7,21. Efter denne beskrivelse, en detaljeret metode til billedbehandling og kvantitativ måling engulfment hjælp konfokal mikroskopi kombineret med 3-dimensionel (3D) overflade volumen rendering vil blive givet. Denne metode er baseret på fast forberedelse væv, men kan også være tilpasset til brug i levende billeddiagnostiske undersøgelser. Vigtigere er det, mens assay er blevet valideret i forbindelse med den sunde, postnatal retinogeniculate system, kan man anvende de samme teknikker til at vurdere andre fagocyt-neuron interaktioner i hele hjernen, og under sygdom samt fagocyt funktion i andre organsystemer.

<table border="0" cellpadding="0" cellspacing="0" width="533">
	<tbody>
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			<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>
	</tbody>
</table>

an engulfment assay: a protocol to assess interactions between cns phagocytes and neurons

Fagocytose er en proces, hvor en celle opsluger materiale (hele cellen, dele af en celle, snavs, etc.) i dens omgivende ekstracellulære miljø og efterfølgende fordøjer dette materiale, almindeligvis gennem lysosomal nedbrydning. Mikroglia er de bosiddende immunceller i det centrale nervesystem (CNS), hvis fagocytotisk funktion er blevet beskrevet i en bred vifte af betingelser fra neurodegenerativ sygdom (fx beta-amyloid clearance i Alzheimers sygdom) til udvikling af sund hjerne (f.eks synaptisk beskæring) 1-6. Følgende protokol er en engulfment assay udviklet til at visualisere og kvantificere microglia-medieret engulfment af præsynaptiske input i udviklingslandene mus retinogeniculate systemet 7. Mens dette assay blev anvendt til at vurdere mikroglia funktion i denne sammenhæng, kan en tilsvarende fremgangsmåde anvendes til at vurdere andre fagocytter i hele hjernen (fx astrocytter) og resten af kroppen(fx perifere makrofager) samt andre sammenhænge, ​​hvor synaptisk remodellering opstår (f.eks hjerneskade / sygdom).

For nylig har vi brugt denne engulfment assay til at visualisere og kvantificere microglia-medieret engulfment af præsynaptiske input i udviklingslandene retinogeniculate (figur 1) 7. RGCS fra CX3CR1-EGFP heterozygote mus blev anterogradely spores med CTB-594 og CTB-647 i det venstre og højre øje, hhv. Efter denne sporing blev EGFP-positive mikroglia i dLGN afbildet. Disse billeder blev efterfølgende overflade-renderet for volumen målinger. Ved at bruge denne...

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 de bosiddende immunceller i centralnervesystemet (CNS) med en høj evne til at fagocytere eller opsluge materiale i deres ekstracellulære miljø. Her er et bredt anvendelig, pålidelig og yderst kvantitativ analyse til at visualisere og måle microglia-medieret engulfment af synaptiske komponenter beskrevet.

En engulfment Assay: En protokol til Vurdere Interaktioner mellem 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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