Name: in vitro phagocytosis of myelin debris by bone marrow-derived macrophages
Uploaded: 2017-12-30T15:00:00
Description: Watch this Scientific Journal Video about In Vitro Phagocytosis of Myelin Debris by Bone Marrow-Derived Macrophages at JoVE.com

Forfatterne vil gerne takke Glenn Sanger-Hodgson, Media Specialist ved FSU College of Medicine for alle hans arbejde i video-produktion, redigering og voice-over.
Dette arbejde blev støttet af National Institutes of Health (R01GM100474 og R01GM072611).

Metoderne beskrevet her og i afsnit 2 er blevet godkendt af Florida State University institutionelle Animal Care og brug udvalg (IACUC) og følger i Guide retningslinjer for pleje og brugen af forsøgsdyr, 8th edition . Alle dyr, der anvendes i dette i denne protokol er hus i en dedikeret laboratorium animalsk facilitet indtil brug. Ingen i vivo eksperimenter blev udført før at ofre. Animalske numre var baseret på eksperimentel behov ved hjælp af gennemsnitlige celle og myelin samlinger s...

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De procedurer, der beskrives her udnytter både frisk isolerede rå CNS myelin debris og primære knoglemarv-afledte makrofager. For at reducere dyrs udgifter, anbefaler vi, at både hjerne og knoglemarv celler høstes fra hver musen på tidspunktet for offer. To forskere, der arbejder sammen kan udarbejde begge materialer samtidig. Alternativt, hjerner kan opbevares ved-80 ° C i PBS suppleret med antibiotika før myelin vragrester isolation. Det har været vores erfaring, at hjerne kan opretholdes i disse betingelser f...

Knoglemarv-afledte makrofager (BMDMs) er et vigtigt bindeled mellem de medfødte og adaptive immunforsvar. Som antigen præsentere celler (PMV'er), de kan kommunikere med lymfocytter via både antigen præsentation og cytokin frigive1,2,3. Men som professionelle fagocytter, deres primære funktion er at rydde patogener, alderen celler og celleaffald1,4. Efter en rygmarvsskade (SCI) genereres betydelige mængder af myelin vragrester fra døende oligodendrocytes, ansvarlig for CNS axon myelination5celletype. Vi og andre har vist, at clearance af myelin vragrester er primært ansvar infiltrerer BMDMs5,6,7. Men inden for rygmarvsskade websteder engulfment af myelin vragrester er blevet foreslået til at flytte disse normalt anti-inflammatoriske celler mod en pro-inflammatoriske tilstand5,8,9. Som centrale mediatorer af neuro-betændelse i såret rygmarven er BMDMs vigtige kliniske mål.
For at hjælpe med at undersøge BMDMs indflydelse i den skadede rygmarven, har vi udviklet en in vitro- model for at direkte studere hvordan BMDMs reagere på myelin debris. For at forbedre biologisk relevans, bruges både primære murine BMDMs og frisk isolerede myelin snavs i disse undersøgelser. Som sådan, de metoder, der præsenteres her også detaljeret isolation og kultur af primære murine BMDMs, og en modificeret saccharose gradient teknik bruges til at isolere murine CNS afledt myelin vragrester10,11,12. Myelin snavs kan let mærket med et fluorescerende farvestof, carboxyfluorescein succinimidyl ester (CFSE), til at spore dens internalisering af BMDMs. CFSE er velegnet til dette program for det er ikke-cytotoksiske, og dens smalle fluorescerende spectrum tilladelser multiplexing med andre fluorescerende sonder13,14. Efter fagocytose, myelin vragrester lipider transporteres gennem lysosomer og pakkede som neutral lipider i intracellulære lipid dråber5. For at kvantificere denne intracellulære lipid ophobning, præsenterer vi en olie røde O (ORO) farvning metode optimeret til kvantitative billedanalyse. Denne enkle farvning metode producerer robuste reproducerbare resultater og kvantificering15. Disse metoder lette undersøgelsen af myelin vragrester fagocytose og lipid opbevaring med begrænset specialiseret udstyr.

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			<td height="41" style="height:42px;width:180px;">DMEM</td>
			<td style="width:149px;">GE Healthcare Life Sciences</td>
			<td style="width:115px;">SH30243.01</td>
			<td style="width:225px;">High glucose with L-glutamine, sodium pyruvate</td>
		</tr>
		<tr height="62">
			<td height="62" style="height:63px;width:180px;">Penicillin-Streptomycin Solution</td>
			<td style="width:149px;">Corning</td>
			<td style="width:115px;">30-002-CI</td>
			<td style="width:225px;">100X Solution</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:42px;width:180px;">New Born Calf Serum (NCS)</td>
			<td style="width:149px;">Rocky Mountain Biologics</td>
			<td style="width:115px;">NBS-BBT-5XM</td>
			<td style="width:225px;">United States Origin</td>
		</tr>
		<tr height="83">
			<td height="83" style="height:83px;width:180px;">NCTC clone 929 [L cell, L-929, derivative of Strain L]&#160;</td>
			<td style="width:149px;">ATCC</td>
			<td style="width:115px;">CCL-1</td>
			<td style="width:225px;">L929 Cell Line of Conditioned Media Preparation</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:42px;width:180px;">24-well Cell Culture Plates</td>
			<td style="width:149px;">VWR</td>
			<td style="width:115px;">10062-896&#160;</td>
			<td style="width:225px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:180px;">Cell Culture Dish</td>
			<td style="width:149px;">Greiner Bio-One</td>
			<td style="width:115px;">639960</td>
			<td style="width:225px;">Polystyrine, 145/20mm</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:42px;width:180px;">CFSE Cell Proliferation Kit</td>
			<td style="width:149px;">Thermo Fisher</td>
			<td style="width:115px;">C34570</td>
			<td style="width:225px;">DMSO for Reconsitution Provided</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:42px;width:180px;">Fluoro-gel with Tris Buffer&#160;</td>
			<td style="width:149px;">Electron Microscopy Sciences</td>
			<td style="width:115px;">17985-11</td>
			<td style="width:225px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:180px;">Oil Red O</td>
			<td style="width:149px;">Sigma Aldrich</td>
			<td style="width:115px;">O0625</td>
			<td style="width:225px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:180px;">Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Materials</td>
			<td style="width:149px;">Company</td>
			<td style="width:115px;">Catalog Number</td>
			<td style="width:225px;">Comments</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:42px;width:180px;">Ultracentrifuge Tubes</td>
			<td style="width:149px;">Beckman Coulter</td>
			<td style="width:115px;">326823</td>
			<td style="width:225px;">Thinwall, Polypropylene, 38.5 mL, 25 x 89 mm</td>
		</tr>
		<tr height="62">
			<td height="62" style="height:63px;width:180px;">SW 32 Ti Ultracentrifuge Rotor</td>
			<td style="width:149px;">Beckman Coulter</td>
			<td style="width:115px;">369650</td>
			<td style="width:225px;">SW 32 Ti Rotor, Swinging Bucket, Titanium</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:42px;width:180px;">Hand Held Rotary Homogenizer</td>
			<td style="width:149px;">Fisher Science</td>
			<td style="width:115px;">08-451-71</td>
			<td style="width:225px;"></td>
		</tr>
	</tbody>
</table>

in vitro phagocytosis of myelin debris by bone marrow-derived macrophages

Knoglemarv-afledte makrofager (BMDMs) er modne leukocytter, der tjener en kritisk fysiologisk rolle som professionelle fagocytter i stand til at rydde en bred vifte af partikler. Normalt BMDMs er begrænset fra centralnervesystemet (CNS), men efter en skade, kan de let infiltrere. Én gang inden for den tilskadekomne CNS væv, BMDMs er den primære type ansvarlig for regnskabsafslutning skade-afledte celleaffald, herunder store mængder lipid rige myelin debris. De neuropatologiske udløbere af BMDM infiltration og myelin vragrester fagocytose inden for CNS er komplekse og ikke godt forstået. De protokoller er beskrevet her, giver mulighed for direkte i in vitro -undersøgelse af BMDMs i forbindelse med CNS skade. Vi dækker murine BMDM isolation og kultur, myelin vragrester forberedelse og assays for at vurdere BMDM myelin vragrester fagocytose. Disse teknikker giver robust kvantificerbare resultater uden behov for betydelige specialiseret udstyr eller materialer, men let kan tilpasses til at imødekomme behovene hos forskere.

Behandling af BMDMs med CFSE mærket myelin vragrester bør give klare internalisering (figur 2). Mens en 3-timers interaktion tid er tilstrækkeligt for BMDMs til at phagocytosis nok tilføjet myelin vragrester for robust downstream påvisning, kan intracellulære ophobning observeres med så lidt som 1 time af interaktion. Dog kan nogle myelin vragrester stadig være til stede på cellens overflade efter vask. Dette kan skyldes utilstrækkelig vask eller partikler ikke at være fuldt inter...

Watch this Scientific Journal Video about In Vitro Phagocytosis of Myelin Debris by Bone Marrow-Derived Macrophages at JoVE.com

In Vitro Phagocytosis of Myelin Debris by Bone Marrow-Derived Macrophages

Vi præsentere metoder til at vurdere den fagocyterende kapacitet af primære murine knoglemarv-afledte makrofager ved hjælp af fluorescently mærket myelin debris og intracellulære lipid droplet farvning.

In Vitro Fagocytose af Myelin resterne af knoglemarv-afledte makrofager

Bone marrow-derived macrophages (BMDMs) are mature leukocytes that serve a critical physiological role as professional phagocytes capable of clearing a ...

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In Vitro Phagocytosis of Myelin Debris by Bone Marrow-Derived Macrophages

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