Name: using rna-interference to investigate the innate immune response in mouse macrophages
Uploaded: 2014-11-03T13:00:00
Description: Watch this Scientific Journal Video about Using RNA-interference to Investigate the Innate Immune Response in Mouse Macrophages at JoVE.com

Thanks to Brad Lackford for assistance optimizing some of the techniques described in this manuscript.

1. Opprettholdelse av makrofagcellelinjer <ol><li> Dyrk RAW264.7 og J774A.1 cellelinjer i DMEM supplert med 10% føtalt bovint serum (FBS) ved 37 ° C i nærvær av 5% CO2. Å bidra til å opprettholde sterilitet, tilsett 1% penicillin / streptomycin (penn / strep) til media (selv om dette er strengt tatt ikke nødvendig). </li><li> Kritisk trinn: Pass på at makrofagene vokser i en sunn tilstand for effektiv transfeksjon og siRNA-indusert genet knockdown. Bruk celler mellom passasjene 3 og...

<ol>
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Tallrike studier har blitt publisert i den enkelte gener har blitt målrettet av siRNA i murine makrofager. Mens lipid-mediert transfeksjon har vært brukt til å levere siRNA til makrofagcellelinjer på individuell basis, er disse fremgangsmåter lider av potensielle virkninger på levedyktighet, begrenset gen knockdown, og variasjon fra genet til genet. Å utvikle mer robuste analyser som kan brukes til å målrette gener i middels eller høy gjennomstrømming mote, vi optimalisert teknikker som bruker Amaxa Nucleofec...

I denne protokollen beskriver vi effektive metoder for å hemme gener i mus makrofagcellelinjer hjelp sirnas og overvåke effekten av disse behandlingene på det medfødte immunresponsen. Disse fremgangsmåtene blir gjennomført i 96-brønners-formatet, slik at for RNAi skjermer i middels- eller høy-throughput måte. Som reaksjon på infeksjon, mennesker montere en umiddelbar medfødte immunrespons og en langsommere, men mer spesifikk adaptiv immunrespons. Denne raske medfødte immunrespons innebærer rekruttering og aktivering av fagocytterende medfødte immunceller inkludert makrofager 1. Klassisk aktiverte makrofager er involvert i akutte inflammatoriske responser og produsere antimikrobielle proteiner og forbindelser, cytokiner og chemokiner, og presentere antigener 2,3. Alternativt kan aktiverte makrofager spiller en rolle i regulering av immunitet, opprettholde toleranse, vevsreparasjon og sårheling 4-8. På grunn av deres brede spekter av funksjonerKan makrofager spiller en rolle i en rekke sykdommer som aterosklerose, artritt, og kreft 9. Således har studiet av makrofager vært et sentralt område av forskning i noen tid i et bredt utvalg av sykdoms felt. Til tross for sin betydning i det medfødte immunrespons, kan makrofager være utfordrende celler å jobbe med. Spesielt er det vanskelig å oppnå effektiv transfeksjon ved hjelp av lipid reagenser i makrofager uten assosiert toksisitet 10,11. Dessuten, selv når siRNA er effektivt leveres til makrofager, kan robustheten av RNAi-induserte genet knockdown ofte være relativt moderat, og kan variere fra genet til genet. For å overvinne disse tekniske utfordringer, har vi optimalisert transfeksjon og knockdown-teknikker 12-16 i to muse makrofagcellelinjer, RAW264.7 17 og J774A.1 18. Denne tilnærmingen bruker Amaxa Nucleofector 96 brønner Shuttle System for transfeksjon; Dette systemetbruker en kombinasjon av spesialiserte reagenser og elektroporering for å transfektere celler i 96-brønners format 19. Etter transfeksjon beskriver vi metoder for å maksimere celle utvinning og levedyktighet og for å maksimere påfølgende siRNA-indusert genet knockdown. For å illustrere nytten av denne tilnærmingen, beskriver vi en protokoll for siRNA levering til disse makrofagcellelinjer, stimulere disse cellene med lipopolysakkarid (LPS), og overvåke den medfødte immunrespons ved nivået for produksjon av flere pro-inflammatoriske cytokiner. Vi gir eksempler data der vi målet Toll-like receptor (TLR) familie, hvis medlemmer forstand LPS og andre patogen assosiert molekylære mønstre (PAMPs), å regulere medfødt immunitet.

<table border="0" cellpadding="0" cellspacing="0" >
	<tbody>
		<tr >
			<td >Amaxa nucleofector 96-well shuttle system</td>
			<td>Lonza</td>
			<td>AAM-1001S</td>
		<td></td></tr>
		<tr >
			<td >Amaxa SF cell line 96-well nucleofector kit</td>
			<td>Lonza</td>
			<td>V4SC-2096</td>
		<td></td></tr>
		<tr >
			<td >RAW264.7 mouse macrophage cell line</td>
			<td>ATCC</td>
			<td>TIB-71</td>
		<td></td></tr>
		<tr >
			<td >J774A.1 mouse macrophage cell line</td>
			<td>ATCC</td>
			<td>TIB-67</td>
		<td></td></tr>
		<tr >
			<td >siGenome smartpool siRNA</td>
			<td>Dharmacon</td>
			<td>varies depending on gene</td>
		<td></td></tr>
		<tr >
			<td >Non-targeting control siRNA pool</td>
			<td>Dharmacon</td>
			<td>D-001206-13-20</td>
		<td></td></tr>
		<tr >
			<td >Block-iT fluorescent oligo for electrooration</td>
			<td>Invitrogen</td>
			<td>13750062</td>
		<td></td></tr>
		<tr >
			<td >Ultrapure E. coli O111:B4 LPS</td>
			<td>List Biological Laboratories</td>
			<td>421</td>
		<td></td></tr>
		<tr >
			<td >DMEM, high glucose</td>
			<td>Invitrogen</td>
			<td>11995-065</td>
		<td></td></tr>
		<tr >
			<td >RPM1-1640</td>
			<td>Invitrogen</td>
			<td>11875-093</td>
		<td></td></tr>
		<tr >
			<td >Penicillin-Streptomycin Solution (Pen/Strep)</td>
			<td>Fisher</td>
			<td>SV30010</td>
		<td></td></tr>
		<tr >
			<td >0.25% Trypsin-EDTA</td>
			<td>Invitrogen</td>
			<td>25200072</td>
		<td></td></tr>
		<tr >
			<td >96 well tissue culture plates</td>
			<td>Fisher</td>
			<td>07-200-89 </td>
		<td></td></tr>
		<tr >
			<td >96 well round bottom sterile plates (not coated)</td>
			<td>Fisher</td>
			<td>07-200-745</td>
		<td></td></tr>
		<tr >
			<td >Mouse IL-6 Duoset ELISA kit</td>
			<td>R&#38;D Biosystems</td>
			<td>DY406</td>
		<td></td></tr>
		<tr >
			<td >Mouse TNFa Duoset ELISA kit</td>
			<td>R&#38;D Biosystems</td>
			<td>DY410</td>
		<td></td></tr>
		<tr >
			<td >Fluorescein diacetate</td>
			<td>Sigma-Aldrich</td>
			<td>F7378</td>
		<td></td></tr>
		<tr >
			<td >RLT Bufer</td>
			<td>Qiagen</td>
			<td>79216</td>
		<td></td></tr>
		<tr >
			<td >Rneasy mini kit</td>
			<td>Qiagen</td>
			<td>74134</td>
		<td></td></tr>
		<tr >
			<td >Vybrant Phagocytosis Assay Kit</td>
			<td>Invitrogen</td>
			<td>V-6694</td>
		<td></td></tr>
	</tbody>
</table>

using rna-interference to investigate the innate immune response in mouse macrophages

Macrophages are key phagocytic innate immune cells. When macrophages encounter a pathogen, they produce antimicrobial proteins and compounds to kill the pathogen, produce various cytokines and chemokines to recruit and stimulate other immune cells, and present antigens to stimulate the adaptive immune response. Thus, being able to efficiently manipulate macrophages with techniques such as RNA-interference (RNAi) is critical to our ability to investigate this important innate immune cell. However, macrophages can be technically challenging to transfect and can exhibit inefficient RNAi-induced gene knockdown. In this protocol, we describe methods to efficiently transfect two mouse macrophage cell lines (RAW264.7 and J774A.1) with siRNA using the Amaxa Nucleofector 96-well Shuttle System and describe procedures to maximize the effect of siRNA on gene knockdown. Moreover, the described methods are adapted to work in 96-well format, allowing for medium and high-throughput studies. To demonstrate the utility of this approach, we describe experiments that utilize RNAi to inhibit genes that regulate lipopolysaccharide (LPS)-induced cytokine production.

For å demonstrere effektiviteten av transfeksjon ved bruk av denne tilnærmingen, overvåket vi opptaket av FITC-merket siRNA ved hjelp av et strømningscytometer (figur 1). For å illustrere nytten av vår tilnærming for overvåking av medfødte immunrespons, vi transfektert sirnas rettet kjente medfødte immun regulatoriske gener inn i RAW264.7 makrofagcellelinje, stimuleres cellene med LPS, og deretter overvåket produksjonen av proinflammatoriske cytokiner IL- 6 og TNF...

Watch this Scientific Journal Video about Using RNA-interference to Investigate the Innate Immune Response in Mouse Macrophages at JoVE.com

Using RNA-interference to Investigate the Innate Immune Response in Mouse Macrophages (Video) | JoVE

In this protocol, we describe methods to efficiently transfect murine macrophage cell lines with siRNAs using the Amaxa Nucleofector 96-well Shuttle System, stimulate the macrophages with lipopolysaccharide, and monitor the effects on inflammatory cytokine production.

Ved hjelp av RNA-interferens for å undersøke det medfødte immunrespons i Musemakrofager

Macrophages are key phagocytic innate immune cells.  When macrophages encounter a pathogen, they produce antimicrobial proteins and compounds to kill the ...

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Immunology and Infection

Using RNA-interference to Investigate the Innate Immune Response in Mouse Macrophages

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