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.

대 식세포 세포주 1. 유지 보수 <ol><li> RAW264.7 성장 및 DMEM에서 J774A.1 세포주 5 % CO 2 존재하에 37 ºC에서 10 % 소 태아 혈청 (FBS)으로 보충. , 불임을 유지하는 데 도움이 (이 반드시 필요한 것은 아니지만) 미디어에 1 % 페니실린 / 스트렙토 마이신 (펜 / 연쇄상 구균)를 추가합니다. </li><li> 중요한 단계 : 대 식세포는 효율적인 형질과의 siRNA에 의한 유전자 최저의 건강한 상태로 성?...

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수많은 연구하는 개별 유전자가 쥐의 대 식세포에 siRNA를 표적이되어 발표되었다. 지질 - 매개 형질 감염이 개별적으로 대 식세포 세포주에 siRNA를 전달하기 위해 사용되었지만, 이러한 방법은 유전자의 유전자 생존 제한된 유전자 녹다운 및 변동에 미치는 잠재적 영향 겪는다. 중간 또는 높은 처리량 방식으로 유전자를 대상으로 사용할 수있는보다 강력한 분석을 개발하기 위해, 우리는 유전자 ?...

이 프로토콜에서, 우리는를 사용하여 siRNA를 마우스 대식 세포주에서 유전자를 억제하고 선천성 면역 반응에 대한 이러한 치료의 효과를 모니터링하는 효율적인 방법을 설명한다. 이러한 절차는 중간 또는 높은 처리량 방식 RNAi의 스크린을 허용 96- 웰 포맷에서 수행된다. 감염에 응답하여, 인간은 즉각적인 선천성 면역 반응과 더 느리지 만 특정 적응성 면역 반응을 탑재. 이 빠른 선천성 면역 반응은 대 식세포 (1)을 포함하여 식세포 선천성 면역 세포의 모집 및 활성화를 포함한다. 클래식 활성화 대 식세포는 급성 염증 반응에 관여하는 항균 단백질과 화합물, 사이토 카인과 케모카인 및 현재 항원 2,3를 생산하고 있습니다. 또는 활성화 된 대 식세포는 허용 오차, 조직 복구를 유지, 면역 조절에 중요한 역할을하고, 4-8 상처 치유. 때문에 기능의 그들의 다양한의대 식세포는 죽상 동맥 경화증, 관절염, 암 (9)을 포함하여 다수의 질병에서 역할을 할 수있다. 따라서, 대 식세포의 연구는 질병 다양한 분야에서 오랫동안 연구의 핵심 영역이었다. 선천성 면역 반응에서 그 중요성에도 불구하고, 대 식세포와 함께 작동하도록 세포를 도전 할 수 있습니다. 특히, 관련된 독성 10,11없이 식세포 지질 시약을 사용하여 형질 전환을 효율적으로 얻는 것이 곤란하다. 또한, siRNA를 효율적 식세포로 전달되는 경우에도, 종종 넉다운 RNAi를 유도 유전자의 견고성 상당히 온건 할 수 있고, 유전자로 유전자로부터 변할 수있다. 이러한 기술적 과제를 극복하기 위해, 우리는 두 마우스 대식 세포주 RAW264.7 17 J774A.1 18 형질 감염 및 녹다운 기술 12-16을 최적화했다. 이 방법은 형질 전환을위한 Amaxa Nucleofector 96 웰 셔틀 시스템을 사용합니다; 이 시스템96- 웰 포맷 (19)에서 세포를 형질 감염 할 특수 시약 및 전기의 조합을 사용한다. 형질 전환에 따라, 우리는 방법 세포 복구 및 생존 능력을 극대화하기 위해 이후의 siRNA에 의한 유전자 최저을 극대화하기 위해 설명합니다. 이 접근 방법의 유용성을 설명하기 위해 리포 폴리 사카 라이드 (LPS)로 이들 세포를 자극하고, 여러 가지 전 염증성 사이토 카인의 생산 수준에서의 선천성 면역 반응을 모니터링, 이들 대 식세포 세포주에 대한 siRNA의 전달을위한 프로토콜을 기술한다. 우리는 우리가 회원 LPS와 다른 병원체 관련 분자 패턴 (PAMPs가)를 감지, 선천성 면역을 조절하는 수신자 같은 수용체 (TLR) 가족을 대상으로하는 샘플 데이터를 제공합니다.

<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.

이 방법을 사용하여 형질 감염 효율을 입증하기 위해, 우리는 플로우 사이토 미터 (도 1)를 사용하여 FITC 표지 된 siRNA에의 흡수를 모니터링. 선천성 면역 반응을 모니터링하는 우리의 접근 방법의 유용성을 설명하기 위해, RAW264.7 대식 세포주에 공지 선천성 면역 조절 유전자를 타겟팅 된 siRNA를 형질 LPS로 세포를 자극 한 다음 - 염증성 사이토 카인의 생산을 모니?...

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.

마우스 대 식세포의 타고난 면역 반응을 조사하기 위해 RNA 간섭을 사용하여

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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Quantification of the Respiratory Burst Response as an Indicator of Innate Immune Health in Zebrafish

Quantification of the Respiratory Burst Response as an Indicator of Innate Immune Health in Zebrafish (Video) | JoVE

제브라 피쉬의 선천성 면역 건강의 지표로 호흡 버스트 응답의 정량화

The phagocyte respiratory burst is part of the innate immune response to  pathogen infection and involves the production of reactive oxygen species (ROS). ...

Dissecting Innate Immune Signaling in Viral Evasion of Cytokine Production

Dissecting Innate Immune Signaling in Viral Evasion of Cytokine Production (Video) | JoVE

사이토 카인 생산의 바이러스 성 회피에 타고난 면역 신호를 해부

In response to a viral infection, the host innate immune response is activated to up-regulate gene expression and production of antiviral cytokines. ...

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection (Video) | JoVE

웨스트 나일 바이러스 돌연변이 균주의 감염에에서 MyD88 매개 세포 면역 반응의 생체 외 분석

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in ...

Non-invasive Imaging of the Innate Immune Response in a Zebrafish Larval Model of Streptococcus iniae Infection

Non-invasive Imaging of the Innate Immune Response in a Zebrafish Larval Model of Streptococcus iniae Infection (Video) | JoVE

의 Zebrafish의 애벌레 모델의 선천성 면역 반응의 비 침습적 영상<em&gt; 연쇄상 구균 iniae</em&gt; 감염

The aquatic pathogen, Streptococcus iniae, is responsible for over 100 million dollars in annual losses for the aquaculture industry and is capable of ...

Using Phylogenetic Analysis to Investigate Eukaryotic Gene Origin

Using Phylogenetic Analysis to Investigate Eukaryotic Gene Origin (Video) | JoVE

사용 하 여 계통 발생 분석은 진 핵 유전자 원산지 조사

Phylogenetic analysis uses nucleotide or amino acid sequences or other parameters, such as domain sequences and three-dimensional structure, to construct ...