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 gjerne takke Glenn Sanger-Hodgson, Media spesialist på FSU College of Medicine for alle sitt arbeid i videoproduksjon, redigering og voice-over.
Dette arbeidet ble støttet av National Institutes of Health (R01GM100474 og R01GM072611).

Metodene beskrevet her og i del 2 har blitt godkjent av Florida State University institusjonelle Animal Care og bruk Committee (IACUC) og følger retningslinjene som er angitt i Guide for omsorg og bruk av forsøksdyr, 8th utgave . Alle dyr som brukes i denne denne protokollen er hus i en dedikert laboratorium dyr anlegget før bruk. Ingen i vivo eksperimentering ble utført før ofre. Dyr tall var basert på eksperimentell behov med gjennomsnittlig cellen og myelin samlinger som en guide for...

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Prosedyrene som er beskrevet her benytter både fersk isolert råolje CNS myelin rusk og primære Ben margtransplantasjon-avledet makrofager. For å redusere dyr utgifter, anbefaler vi at både hjerne og bein margtransplantasjon celler høstes fra hver musen ved offer. To forskere samarbeide kan forberede begge materialene samtidig. Hjernen kan også lagres på-80 ° C i PBS med antibiotika før myelin rusk isolasjon. Det er vår erfaring at hjerner kan opprettholdes under disse forholdene i opptil 1 måned uten merkbar ...

Ben margtransplantasjon-avledet makrofager (BMDMs) er et viktig bindeledd mellom de medfødte og adaptive immunsystem. Som antigen presentere celler (APCs), de kan kommunisere med lymfocytter via både antigen-presentasjon og cytokin slipp1,2,3. Men som profesjonell phagocytes er deres primære funksjon å fjerne patogener, alderen celler og mobilnettet rusk1,4. Etter en ryggmargsskade (SCI), betydelige mengder myelin rusk genereres fra døende oligodendrocytes, hvilken celle ansvarlig for CNS axon myelination5. Vi og andre har vist at klarering av myelin rusk er primært ansvarlig for infiltrere BMDMs5,6,7. Men innen ryggmargsskade har nettsteder engulfment av myelin avfall blitt foreslått for å skifte disse normalt anti-inflammatoriske celler mot en pro-inflammatoriske staten5,8,9. Som nøkkel meglere på Nevro-betennelse i skadet ryggmargen er BMDMs viktig kliniske mål.
For å undersøke påvirkning av BMDMs i skadet ryggmargen, har vi utviklet en i vitro modell å direkte studere hvordan BMDMs svare på myelin rusk. For å forbedre biologiske relevans, brukes både primære murine BMDMs og ferske isolert myelin rusk i disse undersøkelsene. Som sådan, metodene presenteres her også detalj isolasjon og kultur av primære murine BMDMs, og en modifisert sukrose gradient teknikk som brukes til å isolere murine CNS avledet myelin rusk10,11,12. Myelin rusk kan være lett merket med et fluorescerende farge, carboxyfluorescein succinimidyl ester (CFSE), å spore sin internalisering av BMDMs. CFSE er godt egnet for dette programmet fordi det er ikke-cytotoksiske og dens smale fluorescerende spekteret tillater Multiplexing med andre fluorescerende sonder13,14. Etter fagocytose, myelin rusk lipider transporteres gjennom lysosomer og pakket som nøytral lipider i intracellulær lipid dråper5. For å måle denne intracellulær lipid opphopningen, presenterer vi en olje Red O (ORO) flekker metoden optimalisert for kvantitative bildeanalyser. Denne enkle flekker metoden gir robust reproduserbar resultater og kvantifisering15. Disse metodene lette studiet av myelin rusk fagocytose og lipid oppbevaring med begrenset spesialisert utstyr.

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	<tbody>
		<tr height="41">
			<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

Ben margtransplantasjon-avledet makrofager (BMDMs) er modne leukocytter som tjener en kritisk fysiologiske rolle som profesjonell phagocytes i stand til å fjerne en rekke partikler. Vanligvis BMDMs er begrenset fra sentralnervesystemet (CNS), men etter en skade, kan de lett infiltrere. En gang innenfor den skadde CNS vevet, BMDMs er den primære celle typen ansvarlig for rydding av skade-avledet mobilnettet rusk, inkludert store mengder lipid rik myelin rusk. Neuropathological betydningen av BMDM infiltrasjon og myelin rusk fagocytose i CNS er komplekse og ikke godt forstått. Protokollene som beskrevet her, Tillat for direkte i vitro studier av BMDMs i forbindelse med CNS skade. Vi dekker murine BMDM isolasjon og kultur, myelin rusk forberedelser og analyser for å vurdere BMDM myelin rusk fagocytose. Disse teknikkene gir robust målbare resultater uten behov for betydelig spesialisert utstyr eller materialer, men kan enkelt tilpasses for å møte behovene til forskere.

Behandling av BMDMs med CFSE merket myelin rusk skal gi klare internalisering (figur 2). Mens gangen 3 timers samhandling er tilstrekkelig for BMDMs å phagocytose nok ekstra myelin rusk for robust nedstrøms gjenkjenning, kan intracellulær opphopning observeres med så lite som 1 time interaksjon. Men kan noen myelin rusk fortsatt finnes på cellens overflate etter vask. Dette kan skyldes utilstrekkelig vask eller partikler ikke blir fullstendig internalisert i den tidlige fasen av fagocyt...

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 presenterer metoder for å vurdere phagocytic kapasitet primære murine Ben margtransplantasjon-avledet makrofager fluorescently merket myelin rusk og intracellulær lipid slippverktøy flekker.

In Vitro Fagocytose av Myelin rusk av Ben margtransplantasjon-avledet 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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