We would like to thank the entire Lazzaro lab, and especially Susan Rottschaefer, for their help in both reviewing and testing these protocols. This is a product of their cumulative expertise. Work in the Lazzaro lab is supported by grants R01 AI083932 and R01 AI064950 from the US National Institutes of Health.

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None of the authors have competing interests or conflicting interests.

여기에 설명 된 절차는 초파리의 엄격하고 높은 품질의 감염을 산출한다. 도시 된 예는 주로 비던 레트 게리와 E 감염에 초점을 맞추고 대장균하지만 프로토콜 적응력이며 호스트 사육 및 관리 조건의 범위에서 다양한 세균 감염에 적용될 수있다. 최적의 실험 방법의 세부 사항은 감염에 사용되는 세균 숙주의 유전자형, 전반적인 실험 조건에 의존 할 것이다. 그것은 강력하게 새로운 실험 조건 더 야심 찬 프로젝트를 시작하기 전에 파일럿 테스트하는 것이 좋습니다. 좋은 출발점은 100 배의 범위에서 세 감염 량을 테스트하는 것이다. 플라이 100 균체 - 매우 독성이 강한 병원균은 종종 최상의 10 정도의 매우 낮은 감염성 투여 량으로 도입된다. 더 온건 한 병원균은 FL 당 약 1,000 박테리아의 높은 용량으로 주입 될 수있다y 및 비 병원균은 박테리아 플라이 10,000만큼 높은 용량으로 주입 될 수있다. 그것은 세로 시계열상에서 병원균 부하 호스트 사망률 면역계 활성을 추적하여 새로운 감염의 역학을 정의하는 것이 유익하다. 병원체로드 및 호스트 유전자 발현의 측정이 파괴 분석이기 때문에, 그것을 측정 할 때마다 포인트에 대한 실험의 초기에 뚜렷한 파리를 감염시킬 필요가있다. 바늘로 한 번 찌르기 또는 마이크로 모세관 기반 주입을 사용할지 여부를 결정할 때, 이것은 장점과 한계 각 접근법에 존재 주목하는 것이 중요하다. 모세관 주입 모두 적당한 팽압을 증가시키고 염 또는 현탁 또는 담체에 용해 된 다른 분자를 도입 플라이으로 액체의 부피를 도입한다. 모세관 주입은 필요한 장비의 주입 시설 또는 구매에 액세스 할 수 있어야합니다. 정화조 바늘로 한 번 찌르기가 더 특별 장비 및 소개가 필요하지 않습니다무시할 플라이에 용지와는 일반적으로 날아간 다수의 감염에 더 효율적이다. 그러나 바늘로 한 번 찌르기 감염은 모세관 주입 달성 할 수 감염 량을 통해 정밀하게 제어 할 수 없습니다. 본 프로토콜은 기계적 주입량을 조절 사출 장치에 집중하지만, 가압 된 공기의 이산 펄스에 기초하여 주입 시스템도있다. (20, 21)이 일반적으로 장치가 여기에 추천보다 비싸며 각 공기 펄스의 보정을 필요 순서대로 바늘 일관된 주입 볼륨을 확인합니다. 파리는 야생에서 박테리아와 체계적으로 감염 될 방법에 대한 상당한 논쟁하지만 약간의 데이터가 있습니다. 일부 연구자들은 초파리가 병원성 박테리아를 섭취 할 때 자연 감염의 대부분이 발생 박테리아 이후 전신 감염을 설정하기 위해 장을 탈출 할 수있는 멋 부리다. 그러나, FE 매우있다알려진 W 박테리아는 D의 장을 통과 할 수 있도록 melanogaster의,이 기능을 수행하는 사람들은 파리 (22, 23)에 매우 치명적이다. 또 다른 이론은 실패 포식 시도 나 ectoparasitic 진드기의 공격으로부터 탈출을 통해 cuticular 부상을 유지 정기적으로 운항하는 것입니다. 이 가설은 야생 D의 빈번한 수집에 의해 지원됩니다 치유 상처 (게시되지 않은 관찰)을 가리킨다 melanization 명소 베어링 melanogaster의. 진드기 초파리 24 세균 감염을 송신하는 것으로 나타났다 진드기 남긴 상처 이차적 꿀벌 25 세균에 의해 감염 될 수있다. 그러나 D의 중심 진드기 또는 기타 기회 감염의 자연 주파수 표피 침해 통해 melanogaster의는 알려져 있지 않다. 여기에 설명 된 프로토콜은 직접 상피 장벽 또는 행동 면역 글로불린을 무시 정량 주입을 통해 체액에 박테리아의 도입을 허용NITY. D.에 병원성 박테리아를 공급하기위한 방법 이 Vodovar melanogaster의 외. 22 Nehme 외. (23)에 기재되어있다. 많은 곤충 병원성 박테리아는 연구자들이 편안하게 그들과 함께 작업 할 수 있도록, 거의 또는 전혀 인간의 건강에 위험을 초래할. 또한, 소수의 세균 실험의 개입없이 접촉에 초파리를 감염 할 수있는 능력 때문에 오염 된 표면을 통해 실험을 통해 세균 감염의 &quot;전염병&quot;확산의 위험이 있거나 일반적으로 매우 낮은 파리를 탈출했다. 그럼에도 불구하고, 적절한 봉쇄 조치가 탈출 감염된 파리를 방지하기 위해 장소에서 어떤이 파리를 탈출 탈환을 위해 있는지 확인하는 것이 좋습니다. 실험실에서 사용되는 병원균의에 상응하는 생물학적 안전 수준이 장착되어야하며, 미생물학 표준 모범 사례를 채용해야한다. 실험 infecti여기에 설명 된 방법에 임의의 박테리아의 용량을 가진 초파리 melanogaster의 감염 수 있습니다. 감염이 확립되면, 세균 증식이나 간극의 동역학을 측정하는 호스트 사망률을 추적하고, 숙주 면역계의 유도를 분석하기 쉽다. 감염된 초파리 용이하게 형성되거나 또는 감염에 의해 형성되는 생리 기능의 테스트를 포함하는 다른 표현형 분석을 거치게 될 수있다. 설명 된 절차는 저렴 상대적으로 적은 특수 장비를 필요로하고, 쉽게 연구 및 교육 연구소의 폭에 걸쳐 다양한 프로젝트에 사용하기에 순종하고, 알게된다.

과일 초파리 비행은 실험 조작에 매우 의무가 있으며, 넓은을 개발했다 광범위한 과학계에 의해 백업됩니다 기능과 면역 방어. 초파리의 진화 저렴하고 뒤쪽으로 쉽게 공부를위한 최고의 모델 생물 중 하나입니다 연구 도구의 배열. 선천성 면역의 여러 측면은 수신자 같은 수용체와 NF-kB의 가족 전사 인자, JAK / STAT 신호 및 JNK 경로의 반응에 의해 매개 신호 전달을 포함, 곤충, 포유류 사이에 보존되어있다. 1,2 이들 유전자 및 경로 수의 기능 D에 조회 할 melanogaster의 돌연변이를 사용하여 또는 RNAi의 그 증감 경로 활동 knockdowns 3 -. 또한 6 초파리 진화 생활사 이론의 맥락에서 비롯한 감염 질환의 생리적 영향을 연구하기 위해 사용될 수있다 (7).- 9 모든 그러한 연구는, 그러나, 확실하게 정의 된 처리 조건 하에서 실험 파리 감염 능력에 의존한다. 여기에 설명 된 절차는 초파리에 강력하고 반복적 인 세균 감염을 제공하고 이후 감염의 심각도를 측정하고 호스트 면역 반응을 정량화하기위한 방법 론적 프레임 워크를 제공합니다. 초파리는 자연과 실험적으로 세균, 곰팡이, 바이러스, 선충과 포식 기생자 말벌을 포함 기생충과 병원균의 다양한에 감염 될 수있다. 현재의 프로토콜은 전신 세균 감염을 제공에 초점을 맞추고 있습니다. 많은 다른 세균 감염 파리 사용될 수 있으며, 실험자의 선택은 요구되는 정밀한 과학 질문에 기초한다. 예를 들어, 인간 임상 분리가 박테리아 병원성 메카니즘 (10)을 연구하기 위해 이용 될 수 있거나, 생태 학적으로 중요한 균주 preferre 수있다진화 연구를위한 D. 11 일부 박테리아는 D의 유능한 병원균은 melanogaster의, 감염에 증식 및 호스트 질병 또는 사망의 원인. 다른 박테리아를 효과적으로 호스트의 면역 시스템에 의해 관리하고 몇 일 이내에 삭제됩니다. 이 예제에서, 비던 레트 게리 호스트 사망을 야기하고 생존 호스트에 계속. 대장균이 숙주 면역계에 의해 클리어되지 않은 병원체로 사용될 수있는 증식 성 병원체로서 사용될 것이다. 감염은 직접 플라이의 체강 내에 세균의 도입에 의해 확립 될 것이다. 이러한 접근 방식에 관계없이 송신 고유 모드의 전신 감염의 조사를 허용 상피 장벽 보호 동작을 우회. 실험적으로 전신 감염을 설정하기위한 두 가지 주요 방법이 있습니다. 첫 번째에서 nanoinjector하고 뽑아 유리 모세관 바늘의 정확한 수를 주입하는 데 사용됩니다파리에 박테리아. 이 방법은 감염 투여 량의 넓은 동적 범위를 허용하고 매우 정량적으로 반복되는 장점을 갖는다. 두 번째 방법은 오수 바늘로 한 번 찌르기 감염을 제공하는 것입니다. 이러한 접근 방식은 빠른 인 및 특별한 장치를 필요로하지 않는 장점을 갖는다. 감염이 성립되면, 이는 전신 병원체 부하 호스트 사망률 및 유도 성 면역계 활성을 측정하는 것이 가능해진다. 물론, 추가로 임의의 개수의 표현형을 생각할 감염된 D. 측정 될 수있다 후 감염 번식력 (12), 학습 능력 (13), 신진 대사 상태 (14), 또는 상상 할 수 있습니다 거의 모든 다른 특성을 포함 melanogaster의,.

<table border="0" cellpadding="0" cellspacing="0" width="815">
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		<tr height="21">
			<td height="21" style="height:21px;width:399px;">10mL Syringe</td>
			<td style="width:239px;">BD</td>
			<td style="width:177px;">309604</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mineral Oil, White, light</td>
			<td>Macron Fine Chemicals</td>
			<td>6358-10</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">minutein pins</td>
			<td style="width:239px;">Fine Science Tools</td>
			<td style="width:177px;">26002-10</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">1.5mL&#160; Microcentrifuge tubes; Seal Rite</td>
			<td style="width:239px;">USA Scientific Inc.</td>
			<td style="width:177px;">1615-5500</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">Motorized Pestle; Talboys Laboratory Stirrer</td>
			<td style="width:239px;">Troemner</td>
			<td style="width:177px;">103</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">Talboys High Throughput Homogenizer</td>
			<td style="width:239px;">OPS Diagnostics</td>
			<td style="width:177px;">930145</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">5/32&#39;&#39; Grinding Balls</td>
			<td>OPS Diagnostics</td>
			<td style="width:177px;">GBSS 156-5000-01</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">Vortex Genie</td>
			<td style="width:239px;">Scientific indurstries inc.</td>
			<td style="width:177px;">G560</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">Multichannel Pipettor (10uL-300uL)</td>
			<td style="width:239px;">Sartorius</td>
			<td>730360</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;"></td>
			<td style="width:239px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">WASP2 Whitley Automated Spiral Plater</td>
			<td style="width:239px;">Microbiology International</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:399px;">ProtoCOL automated colony counter / plate counter/ plate reader&#160;</td>
			<td style="width:239px;">Microbiology International</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">TRIzol</td>
			<td style="width:239px;">Life Technologies</td>
			<td style="width:177px;">15596-026</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">qPCR tubes; Low-Profile 0.2 ml 8-Tube Strips</td>
			<td style="width:239px;">Bio-Rad</td>
			<td style="width:177px;">TLS0801</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">qPCR caps; Optical Flat 8-Cap Strips</td>
			<td style="width:239px;">Bio-Rad</td>
			<td style="width:177px;">TCS0803</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">RQ1 RNase-Free DNase</td>
			<td style="width:239px;">Promega</td>
			<td style="width:177px;">m610a</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">M-MLV Reverse Transcriptase</td>
			<td style="width:239px;">Promega</td>
			<td style="width:177px;">m170b</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">dNTPs</td>
			<td style="width:239px;">Promega</td>
			<td style="width:177px;">U1240</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">Oligo-dT</td>
			<td style="width:239px;">IDT</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">&#160;SsoAdvanced SYBR Green Supermix</td>
			<td style="width:239px;">Bio-Rad</td>
			<td style="width:177px;">172-5260</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CFX Connect Real-Time PCR Detection System</td>
			<td style="width:239px;">Bio-Rad</td>
			<td style="width:177px;">185-5200</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RNasin Ribonuclease Inhibitor</td>
			<td>Promega</td>
			<td>N2115</td>
		</tr>
	</tbody>
</table>

systemic bacterial infection and immune defense phenotypes in drosophila melanogaster

열매는 초파리 melanogaster의 면역 방어의 기능과 진화 연구를위한 최고의 모델 생물 중 하나입니다 비행. 선천성 면역의 여러 측면은 곤충 및 포유 동물 사이에서 보존되고, 초파리 용이 유전자 및 실험적으로 조작 할 수 있기 때문에, 이들은 면역계 기능과 질병의 생리적 영향을 연구하기위한 강력하다. 여기에 입증 절차는 상피 장벽과 방어의 수동적 인 형태를 무시하고 전신 감염에 초점을 허용, 직접 체강에 박테리아의 도입에 의해 파리의 감염을 수 있습니다. 절차는 호스트 사망, 전신 병원체 부하와 호스트 면역 체계의 유도 정도의 측정 속도에 대한 프로토콜을 포함한다. 감염이 절차는 저렴하고 튼튼하고 정량적으로 반복하고, 기능적 유전학, 진화 생활사 및 생리학 연구에 사용될 수있다.

이 섹션은 1 감염 투여 량은 주사에 사용되는 박테리아 현탁액의 광학 밀도 변화 것을 보여준다. 초파리의 세균 감염 후에 얻을 수있다 결과를 도시하고, 확실하게 균질화 및 도금에 의해 추정 될 수 전달 용량은 주사 후 곧바로 날아 . 도 2에 도시 된 바와 같이, 다른 병원체는 용량 의존적 (도 2B)가 될 수있는 호스트 사망률 (도 2A) 및 호스트 사망률 상이한 레벨을 일으킬 수있다. 중요한 것은,이 프로토콜은 감염의 다른 유형이 달성 될 수 있습니다 : 프로 비던 시아 레트 게리 20 일 이상 (그림 3A) 동안 지속 만성, 하위 치명적인 감염을 일으킬 수 있습니다. 그러나 Escerichia 대장균 같은 다른 박테리아는 대부분 육시간 후 감염 (그림 3B)에서 호스트 플라이에 의해 삭제됩니다. 면역 SYS의 유도TEM은 항균 펩티드 사체 (도 4A 및 B)의 RNA 분리 및 후속 QRT-PCR을 통해 추정 할 수있다. 유사하지만 덜 정량적, 항균 펩타이드 유전자 프로모터의 조절하에 GFP를 발현하는 면역 시스템 (도 4C)의 유도를 가시화하는 데 사용될 수 날아간다. <img alt="그림 1" src="/files/ftp_upload/52613/52613fig1.jpg" /> . 그림 1 : 감염성 투여 량을 결정 파리 광학 밀도 (0.0001-0.05)의 범위를 커버하는 세균 현탁액의 50 NL 주사 하였다. 파리 바로 균질화되었고 주입에 의해 도입 된 박테리아의 수를 결정하는 도금. 초기 박테리아 하중이 강하게 초기 OD 주입과 상관 (R 2 = 0.96) <img alt="그림 2" src="/files/ftp_upload/52613/52613fig2.jpg" /> <strong&gt; 그림 2 :. 생존 세균성 병원체에 주입 후는 5 ~ 7 일 이전 남성은 세균 정지 또는 멸균 된 매체의 50 NL 주사와 생존을 위해 관찰 하였다. (A) 야생형 날아간 세 종 중 하나로부터 약 5,000 박테리아 주사 하였다. 호스트 사망률의 비율은 감염에 사용되는 박테리아 종에 크게 의존한다. 50, 500, 5,000 및 플라이 박테리아 - (B) 야생형 초파리는 Enterococcous 칼리스 세 가지 투여 량으로 주사 하였다. 면역계 돌연변이 및 야생형 동종 제어 라인은 약 500 부르크 홀데 리아 cepacia 주사 하였다 높은 감염성 투여 량 (C)로 감염된 경우 파리는 더 빨리 죽지. 페어 로그 - 순위 비교 야생형 비행 돌연변이보다 훨씬 더 잘 감염 (χ 2 = 59.02, DF = 1, P &lt;0.0001) 남아 있음을 보여준다. 그림 3 :. 사출 파리 후 세균 하중은 약 (A) 5,000 피 주사 하였다 레트 게리 (B) 3400 E. 대장균. 세균 부하는 주사 후 즉시 후속 다양한 시점에서 측정되었다. 각 데이터 포인트는 하나의 플라이의 박테리아 하중을 나타낸다. E.을 대장균 신속하게 피하면서 도전 호스트에서 지워집니다 레트 게리는 호스트의 삶의 나머지를 위해 계속 ,. <img alt="그림 4" src="/files/ftp_upload/52613/52613fig4.jpg" /> 그림 4 : 주사 후 면역 유전자 발현의 유도. (A)는 파리 P. 50 NL 주사했다 복부 또는 흉부 중 하나에 레트 게리 또는 멸균 PBS, 또는 CO 제외하고 조작되지 남았다 <sUB는&gt; 2 마취. 여섯 시간 감염 후에, 파리는 유전자 QRT-PCR을 사용하여 측정 하였다 Diptericin RNA의 분리 및 발현을 위해 수집 하였다. (A) 발현 수준은 Diptericin의 비로 rp49 사체에 전사를 그래프 화하고, N (CO 2 컨트롤 바는 각각의 상태로부터 비의 평균 및 표준 오차를 나타낸다 (1)의 발현 수준을 가지는 것으로 정의되도록 스케일링 = 4). (B) 발현 수준은 표준 유도되지 않은 상태로 사용 CO 2 aesthesia 제어 파리에서 평균 C T 값이 ΔΔCT 그래프로된다. 막대는 각 조건에서 ΔΔC T의 평균과 표준 오차를 나타낸다 (N = 4). 주입으로 인해 사이트의 전사 유도의 차이 없지만, 패널 및 B의 비교 ΔΔC T 방식은 잠재적으로 유도 수준을 과대 평가하는 방법을 보여준다. (C)는 프로모터 P. 50 NL 주사 하였다 Diptericin의 통제하에 GFP를 발현 날아간 레트 게리 (OD = 0.1) 후 7 일 후에 군데. GFP 패널은 Diptericin의 활성화 세균에 감염된 파리에서 발기인하지만 미디어 주입 컨트롤을 나타내는 감염된 파리의 복부 지방 본문에 GFP의 발현을 보여줍니다. <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td> 유전자 </td><td> 앞으로 </td><td> 역 </td></tr><tr><td> rp49 (또한 rpL32라고도 함) </td><td> 5 &#39;AGGCCCAAGATCGTGAAGAA 3&#39; </td><td> 5 &#39;GACGCACTCTGTTGTCGATACC 3&#39; </td></tr><tr><td> Diptericin </td><td> 5 &#39;GCGGCGATGGTTTTGG 3&#39; </td><td> 5 &#39;CGCTGGTCCACACCTTCTG 3&#39; </td></tr><tr><td> Drosomycin </td><td> 5 &#39;CTGCCTGTCCGGAAGATACAA 3 &#39; </td><td> 5 &#39;TCCCTCCTCCTTGCACACA 3&#39; </td></tr><tr><td> Defensin </td><td> 5 &#39;GAGGATCATGTCCTGGTGCAT 3&#39; </td><td> 5 &#39;TCGCTTCTGGCGGCTATG 3&#39; </td></tr><tr><td> Attacin </td><td> 5 &#39;CGTTTGGATCTGACCAAGG 3&#39; </td><td> 5 &#39;AAAGTTCCGCCAGGTGTGAC 3&#39; </td></tr><tr><td> Metchnikowan </td><td> 5 &#39;AACTTAATCTTGGAGCGATTTTTCTG 3&#39; </td><td> 5 &#39;ACGGCCTCGTATCGAAAATG 3&#39; </td></tr></tbody></table> 표 1 : QRT-PCR 프라이머.

Watch this Scientific Journal Video about Systemic Bacterial Infection and Immune Defense Phenotypes in Drosophila Melanogaster at JoVE.com

Systemic Bacterial Infection and Immune Defense Phenotypes in Drosophila Melanogaster

Drosophila melanogaster is an outstanding model organism for studying innate immune systems and the physiological consequences of infection and disease. This protocol describes how to deliver robust and quantitatively repeatable bacterial infections to D. melanogaster, and how to subsequently measure infection severity and quantify the host immune response.

전신 세균 감염 및 면역 방어 표현형에<em&gt; 초파리 melanogaster의</em

The fruit fly Drosophila melanogaster is one of the premier model organisms for studying the function and evolution of immune defense. Many aspects of ...

systemic-bacterial-infection-immune-defense-phenotypes-drosophila

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JoVE Journal

Immunology and Infection

Systemic Bacterial Infection and Immune Defense Phenotypes in Drosophila Melanogaster

24.4K 조회수.  Cornell University. Drosophila melanogaster is an outstanding model organism for studying innate immune systems and the physiological consequences of infection and disease. This protocol describes how to deliver robust and quantitatively repeatable bacterial infections to D. melanogaster, and how to subsequently measure infection severity and quantify the host immune response.

비디오: Systemic Bacterial Infection and Immune Defense Phenotypes in Drosophila Melanogaster

Seven Steps to Stellate Cells

Hepatic stellate cells are liver-resident cells of star-like morphology and are located in the space of Disse between liver sinusoidal endothelial cells and hepatocytes1,2. Stellate cells are derived from bone marrow precursors and store up to 80% of the total body vitamin A1, 2. Upon activation, stellate cells differentiate into myofibroblasts to produce extracellular matrix, thus contributing to liver fibrosis3. Based on their ability to contract, myofibroblastic stellate cells can regulate the vascular tone associated with portal hypertension4. Recently, we demonstrated that hepatic stellate cells are potent antigen presenting cells and can activate NKT cells as well as conventional T lymphocytes5. 

 Here we present a method for the efficient preparation of hepatic stellate cells from mouse liver. Due to their perisinusoidal localization, the isolation of hepatic stellate cells is a multi-step process. In order to render stellate cells accessible to isolation from the space of Disse, mouse livers are perfused in situ with the digestive enzymes Pronase E and Collagenase P. Following perfusion, the liver tissue is subjected to additional enzymatic treatment with Pronase E and Collagenase P in vitro. Subsequently, the method takes advantage of the massive amount of vitamin A-storing lipid droplets in hepatic stellate cells. This feature allows the separation of stellate cells from other hepatic cell types by centrifugation on an 8% Nycodenz gradient. The protocol described here yields a highly pure and homogenous population of stellate cells. Purity of preparations can be assessed by staining for the marker molecule glial fibrillary acidic protein (GFAP), prior to analysis by fluorescence microscopy or flow cytometry. Further, light microscopy reveals the unique appearance of star-shaped hepatic stellate cells that harbor high amounts of lipid droplets. 


 Taken together, we present a detailed protocol for the efficient isolation of hepatic stellate cells, including representative images of their morphological appearance and GFAP expression that help to define the stellate cell entity.

Here we describe a method for the isolation of hepatic stellate cells from mouse liver. For stellate cell purification, mouse livers are digested in situ and in vitro by pronase-collagenase treatment prior to density gradient centrifugation. This technique yields highly pure hepatic stellate cells.

셀을 별 모양으로 일곱 단계

Hepatic stellate cells are liver-resident  cells of star-like morphology and are located in the space of Disse between  liver sinusoidal endothelial cells ...

연구

면역학 및 감염병학

Measuring Bacterial Load and Immune Responses in Mice Infected with Listeria monocytogenes

Listeria monocytogenes (Listeria) is a Gram-positive facultative intracellular pathogen1. Mouse studies typically employ intravenous injection of Listeria, which results in systemic infection2. After injection, Listeria quickly disseminates to the spleen and liver due to uptake by CD8&alpha;+ dendritic cells and Kupffer cells3,4. Once phagocytosed, various bacterial proteins enable Listeria to escape the phagosome, survive within the cytosol, and infect neighboring cells5. During the first three days of infection, different innate immune cells (e.g. monocytes, neutrophils, NK cells, dendritic cells) mediate bactericidal mechanisms that minimize Listeria proliferation. CD8+ T cells are subsequently recruited and responsible for the eventual clearance of Listeria from the host, typically within 10 days of infection6.
Successful clearance of Listeria from infected mice depends on the appropriate onset of host immune responses6	. There is a broad range of sensitivities amongst inbred mouse strains7,8. Generally, mice with increased susceptibility to Listeria infection are less able to control bacterial proliferation, demonstrating increased bacterial load and/or delayed clearance compared to resistant mice. Genetic studies, including linkage analyses and knockout mouse strains, have identified various genes for which sequence variation affects host responses to Listeria infection6,8-14. Determination and comparison of infection kinetics between different mouse strains is therefore an important method for identifying host genetic factors that contribute to immune responses against Listeria. Comparison of host responses to different Listeria strains is also an effective way to identify bacterial virulence factors that may serve as potential targets for antibiotic therapy or vaccine design.
We describe here a straightforward method for measuring bacterial load (colony forming units [CFU] per tissue) and preparing single-cell suspensions of the liver and spleen for FACS analysis of immune responses in Listeria-infected mice. This method is particularly useful for initial characterization of Listeria infection in novel mouse strains, as well as comparison of immune responses between different mouse strains infected with Listeria. We use the Listeria monocytogenes EGD strain15 that, when cultured on blood agar, exhibits a characteristic halo zone around each colony due to &beta;-hemolysis1 (Figure 1). Bacterial load and immune responses can be determined at any time-point after infection by culturing tissue homogenate on blood agar plates and preparing tissue cell suspensions for FACS analysis using the protocols described below. We would note that individuals who are immunocompromised or pregnant should not handle Listeria, and the relevant institutional biosafety committee and animal facility management should be consulted before work commences.

Measuring Bacterial Load and Immune Responses in Mice Infected with Listeria monocytogenes

Listeria monocytogenes is a model organism for studying immune responses and genetic susceptibility to intracellular bacteria in mice. This method enables one to measure bacterial load and generate single-cell suspensions of the liver and spleen from mice for FACS analysis to determine changes in immune cells due to Listeria infection.

감염된 마우스에 세균 하중과 면역 응답 측정 리스테리아 monocytogenes</em

Listeria monocytogenes (Listeria) is a Gram-positive facultative intracellular pathogen1. Mouse studies typically employ intravenous injection of ...

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

세균성 접착 연구에 전단 응력을 소개합니다

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

An Introduction to Parasitic Wasps of Drosophila and the Antiparasite Immune Response

Most known parasitoid wasp species attack the larval or pupal stages of Drosophila. While Trichopria drosophilae infect the pupal stages of the host (Fig. 1A-C), females of the genus Leptopilina (Fig. 1D, 1F, 1G) and Ganaspis (Fig. 1E) attack the larval stages. We use these parasites to study the molecular basis of a biological arms race. Parasitic wasps have tremendous value as biocontrol agents. Most of them carry virulence and other factors that modify host physiology and immunity. Analysis of Drosophila wasps is providing insights into how species-specific interactions shape the genetic structures of natural communities. These studies also serve as a model for understanding the hosts' immune physiology and how coordinated immune reactions are thwarted by this class of parasites.
The larval/pupal cuticle serves as the first line of defense. The wasp ovipositor is a sharp needle-like structure that efficiently delivers eggs into the host hemocoel. Oviposition is followed by a wound healing reaction at the cuticle (Fig. 1C, arrowheads). Some wasps can insert two or more eggs into the same host, although the development of only one egg succeeds. Supernumerary eggs or developing larvae are eliminated by a process that is not yet understood. These wasps are therefore referred to as solitary parasitoids.
Depending on the fly strain and the wasp species, the wasp egg has one of two fates. It is either encapsulated, so that its development is blocked (host emerges; Fig. 2 left); or the wasp egg hatches, develops, molts, and grows into an adult (wasp emerges; Fig. 2 right). L. heterotoma is one of the best-studied species of Drosophila parasitic wasps. It is a "generalist," which means that it can utilize most Drosophila species as hosts1. L. heterotoma and L. victoriae are sister species and they produce virus-like particles that actively interfere with the encapsulation response2. Unlike L. heterotoma, L. boulardi is a specialist parasite and the range of Drosophila species it utilizes is relatively limited1. Strains of L. boulardi also produce virus-like particles3 although they differ significantly in their ability to succeed on D. melanogaster1. Some of these L. boulardi strains are difficult to grow on D. melanogaster1 as the fly host frequently succeeds in encapsulating their eggs. Thus, it is important to have the knowledge of both partners in specific experimental protocols.
In addition to barrier tissues (cuticle, gut and trachea), Drosophila larvae have systemic cellular and humoral immune responses that arise from functions of blood cells and the fat body, respectively. Oviposition by L. boulardi activates both immune arms1,4. Blood cells are found in circulation, in sessile populations under the segmented cuticle, and in the lymph gland. The lymph gland is a small hematopoietic organ on the dorsal side of the larva. Clusters of hematopoietic cells, called lobes, are arranged segmentally in pairs along the dorsal vessel that runs along the anterior-posterior axis of the animal (Fig. 3A). The fat body is a large multifunctional organ (Fig. 3B). It secretes antimicrobial peptides in response to microbial and metazoan infections.
Wasp infection activates immune signaling (Fig. 4)4. At the cellular level, it triggers division and differentiation of blood cells. In self defense, aggregates and capsules develop in the hemocoel of infected animals (Fig. 5)5,6. Activated blood cells migrate toward the wasp egg (or wasp larva) and begin to form a capsule around it (Fig. 5A-F). Some blood cells aggregate to form nodules (Fig. 5G-H). Careful analysis reveals that wasp infection induces the anterior-most lymph gland lobes to disperse at their peripheries (Fig. 6C, D).
We present representative data with Toll signal transduction pathway components Dorsal and Sp&auml;tzle (Figs. 4,5,7), and its target Drosomycin (Fig. 6), to illustrate how specific changes in the lymph gland and hemocoel can be studied after wasp infection. The dissection protocols described here also yield the wasp eggs (or developing stages of wasps) from the host hemolymph (Fig. 8).

An Introduction to Parasitic Wasps of Drosophila and the Antiparasite Immune Response

Parasitoid (parasitic) wasps constitute a major class of natural enemies of many insects including Drosophila melanogaster. We will introduce the techniques to propagate these parasites in Drosophila spp. and demonstrate how to analyze their effects on immune tissues of Drosophila larvae.

의 기생충 말벌에 대한 소개<em&gt; Drosophila</em&gt; 그리고 Antiparasite 면역 반응

Most known parasitoid wasp species attack the larval or pupal stages of Drosophila. While Trichopria drosophilae infect the pupal stages of the host (Fig. ...

Flow Cytometric Isolation of Primary Murine Type II Alveolar Epithelial Cells for Functional and Molecular Studies

Throughout the last years, the contribution of alveolar type II epithelial cells (AECII) to various aspects of immune regulation in the lung has been increasingly recognized. AECII have been shown to participate in cytokine production in inflamed airways and to even act as antigen-presenting cells in both infection and T-cell mediated autoimmunity 1-8. Therefore, they are especially interesting also in clinical contexts such as airway hyper-reactivity to foreign and self-antigens as well as infections that directly or indirectly target AECII. However, our understanding of the detailed immunologic functions served by alveolar type II epithelial cells in the healthy lung as well as in inflammation remains fragmentary. Many studies regarding AECII function are performed using mouse or human alveolar epithelial cell lines 9-12. Working with cell lines certainly offers a range of benefits, such as the availability of large numbers of cells for extensive analyses. However, we believe the use of primary murine AECII allows a better understanding of the role of this cell type in complex processes like infection or autoimmune inflammation. Primary murine AECII can be isolated directly from animals suffering from such respiratory conditions, meaning they have been subject to all additional extrinsic factors playing a role in the analyzed setting. As an example, viable AECII can be isolated from mice intranasally infected with influenza A virus, which primarily targets these cells for replication 13. Importantly, through ex vivo infection of AECII isolated from healthy mice, studies of the cellular responses mounted upon infection can be further extended.
Our protocol for the isolation of primary murine AECII is based on enzymatic digestion of the mouse lung followed by labeling of the resulting cell suspension with antibodies specific for CD11c, CD11b, F4/80, CD19, CD45 and CD16/CD32. Granular AECII are then identified as the unlabeled and sideward scatter high (SSChigh) cell population and are separated by fluorescence activated cell sorting 3.
In comparison to alternative methods of isolating primary epithelial cells from mouse lungs, our protocol for flow cytometric isolation of AECII by negative selection yields untouched, highly viable and pure AECII in relatively short time. Additionally, and in contrast to conventional methods of isolation by panning and depletion of lymphocytes via binding of antibody-coupled magnetic beads 14, 15, flow cytometric cell-sorting allows discrimination by means of cell size and granularity. Given that instrumentation for flow cytometric cell sorting is available, the described procedure can be applied at relatively low costs. Next to standard antibodies and enzymes for lung disintegration, no additional reagents such as magnetic beads are required. The isolated cells are suitable for a wide range of functional and molecular studies, which include in vitro culture and T-cell stimulation assays as well as transcriptome, proteome or secretome analyses 3, 4.

We describe the rapid isolation of primary murine type II alveolar epithelial cells (AECII) by flow cytometric negative selection. These AECII show high viability and purity and are suitable for a wide range of functional and molecular studies regarding their role in respiratory conditions such as autoimmune or infectious diseases.

기능 및 분자 연구 기본 손쥐 유형 II 폐포 상피 세포의 흐름 Cytometric 절연

Throughout  the last years, the contribution of alveolar type II epithelial cells (AECII)  to various aspects of immune regulation in the lung has been ...

Quantitative Measurement of the Immune Response and Sleep in Drosophila

A complex interaction between the immune response and host behavior has been described in a wide range of species. Excess sleep, in particular, is known to occur as a response to infection in mammals 1 and has also recently been described in Drosophila melanogaster2. It is generally accepted that sleep is beneficial to the host during an infection and that it is important for the maintenance of a robust immune system3,4. However, experimental evidence that supports this hypothesis is limited4, and the function of excess sleep during an immune response remains unclear. We have used a multidisciplinary approach to address this complex problem, and have conducted studies in the simple genetic model system, the fruitfly Drosophila melanogaster. We use a standard assay for measuring locomotor behavior and sleep in flies, and demonstrate how this assay is used to measure behavior in flies infected with a pathogenic strain of bacteria. This assay is also useful for monitoring the duration of survival in individual flies during an infection. Additional measures of immune function include the ability of flies to clear an infection and the activation of NF&kappa;B, a key transcription factor that is central to the innate immune response in Drosophila. Both survival outcome and bacterial clearance during infection together are indicators of resistance and tolerance to infection. Resistance refers to the ability of flies to clear an infection, while tolerance is defined as the ability of the host to limit damage from an infection and thereby survive despite high levels of pathogen within the system5. Real-time monitoring of NF&kappa;B activity during infection provides insight into a molecular mechanism of survival during infection. The use of Drosophila in these straightforward assays facilitates the genetic and molecular analyses of sleep and the immune response and how these two complex systems are reciprocally influenced.

Quantitative Measurement of the Immune Response and Sleep in Drosophila

To understand a link between the immune response and behavior, we describe a method to measure locomotor behavior in Drosophila during bacterial infection as well as the ability of flies to mount an immune response by monitoring survival, bacterial load, and real-time activity of a key regulator of innate immunity, NF&kappa;B.

면역 반응과 수면에서의 양적 측정<em&gt; Drosophila</em

A complex interaction between the immune response and  host behavior has been described in a wide range of species. Excess sleep, in  particular, is known ...

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence factors, which can subvert and manipulate key host functions. The study of host/pathogen interactions is therefore extremely important to understand bacterial infections and develop alternative strategies to counter infectious diseases. This approach however, requires the development of new high-throughput assays for the unbiased, automated identification and characterization of bacterial virulence determinants. Here, we describe a method for the generation of a GFP-tagged mutant library by transposon mutagenesis and the development of high-content screening approaches for the simultaneous identification of multiple transposon-associated phenotypes. Our working model is the intracellular bacterial pathogen Coxiellaburnetii, the etiological agent of the zoonosis Q fever, which is associated with severe outbreaks with a consequent health and economic burden. The obligate intracellular nature of this pathogen has, until recently, severely hampered the identification of bacterial factors involved in host pathogen interactions, making of Coxiella the ideal model for the implementation of high-throughput/high-content approaches.

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Coxiella burnetii is an obligate intracellular Gram-negative bacterium responsible for the zoonotic disease Q fever. Here we describe methods for the generation of Coxiella fluorescent transposon mutants as well as the automated identification and analysis of the resulting internalization, replication and cytotoxic phenotypes.

세대와의 멀티 표현형 높은 콘텐츠 상영<em&gt; Coxiella burnetii의</em&gt; 트랜스포존 돌연변이

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence ...

Bacterial Leaf Infiltration Assay for Fine Characterization of Plant Defense Responses using the Arabidopsis thaliana-Pseudomonas syringae Pathosystem

In the absence of specialized mobile immune cells, plants utilize their localized programmed cell death and Systemic Acquired Resistance to defend themselves against pathogen attack. The contribution of a specific Arabidopsis gene to the overall plant immune response can be specifically and quantitatively assessed by assaying the pathogen growth within the infected tissue. For over three decades, the hemibiotrophic bacterium Pseudomonas syringae pv. maculicola ES4326 (Psm ES4326) has been widely applied as the model pathogen to investigate the molecular mechanisms underlying the Arabidopsis immune response. To deliver pathogens into the leaf tissue, multiple inoculation methods have been established, e.g., syringe infiltration, dip inoculation, spray, vacuum infiltration, and flood inoculation. The following protocol describes an optimized syringe infiltration method to deliver virulent Psm ES4326 into leaves of adult soil-grown Arabidopsis plants and accurately screen for enhanced disease susceptibility (EDS) towards this pathogen. In addition, this protocol can be supplemented with multiple pre-treatments to further dissect specific immune defects within different layers of plant defense, including Salicylic Acid (SA)-Triggered Immunity (STI) and MAMP-Triggered Immunity (MTI).

Bacterial Leaf Infiltration Assay for Fine Characterization of Plant Defense Responses using the Arabidopsis thaliana-Pseudomonas syringae Pathosystem

Quantification of pathogen growth is a powerful tool to characterize various Arabidopsis thaliana (hereafter: Arabidopsis) immune responses. The method described here presents an optimized syringe infiltration assay to quantify the Pseudomonas syringae pv. maculicola ES4326 growth in adult Arabidopsis leaves.

을 사용해서 식물 방어 응답의 미세 특성에 대한 세균성 잎 침투 분석<em&gt; 애기 장대-슈도모나스 시린 개 (Pseudomonas syringae)</em&gt; Pathosystem

In the absence of specialized mobile immune cells, plants utilize their localized programmed cell death and Systemic Acquired Resistance to defend ...

Extraction of Hemocytes from Drosophila melanogaster Larvae for Microbial Infection and Analysis

During the pathogenic infection of Drosophila melanogaster, hemocytes play an important role in the immune response throughout the infection. Thus, the goal of this protocol is to develop a method to visualize the pathogen invasion in a specific immune compartment of flies, namely hemocytes. Using the method presented here, up to 3 &#215; 106 live hemocytes can be obtained from 200 Drosophila 3rd instar larvae in 30 min for ex vivo infection. Alternatively, hemocytes can be infected in vivo through injection of 3rd instar larvae followed by hemocyte extraction up to 24 h post-infection. These infected primary cells were fixed, stained, and imaged using confocal microscopy. Then, 3D representations were generated from the images to definitively show pathogen invasion. Additionally, high-quality RNA for qRT-PCR can be obtained for the detection of pathogen mRNA following infection, and sufficient protein can be extracted from these cells for Western blot analysis. Taken together, we present a method for definite reconciliation of pathogen invasion and confirmation of infection using bacterial and viral pathogen types and an efficient method for hemocyte extraction to obtain enough live hemocytes from Drosophila larvae for ex vivo and in vivo infection experiments.

Extraction of Hemocytes from Drosophila melanogaster Larvae for Microbial Infection and Analysis

This method demonstrates how to visualize pathogen invasion into insect cells with three-dimensional (3D) models. Hemocytes from Drosophila larvae were infected with viral or bacterial pathogens, either ex vivo or in vivo. Infected hemocytes were then fixed and stained for imaging with a confocal microscope and subsequent 3D cellular reconstruction.

미생물 감염 및 분석에 대 한 초파리 melanogaster 애벌레에서 Hemocytes의 추출

During the pathogenic infection of Drosophila melanogaster, hemocytes play an important role in the immune response throughout the infection. Thus, the ...

Establishment of Viral Infection and Analysis of Host-Virus Interaction in Drosophila Melanogaster

Virus spreading is a major cause of epidemic diseases. Thus, understanding the interaction between the virus and the host is very important to extend our knowledge of prevention and treatment of viral infection. The fruit fly Drosophila melanogaster has proven to be one of the most efficient and productive model organisms to screen for antiviral factors and investigate virus-host interaction, due to powerful genetic tools and highly conserved innate immune signaling pathways. The procedure described here demonstrates a nano-injection method to establish viral infection and induce systemic antiviral responses in adult flies. The precise control of the viral injection dose in this method enables high experimental reproducibility. Protocols described in this study include the preparation of flies and the virus, the injection method, survival rate analysis, the virus load measurement, and an antiviral pathway assessment. The influence effects of viral infection by the flies' background were mentioned here. This infection method is easy to perform and quantitatively repeatable; it can be applied to screen for host/viral factors involved in virus-host interaction and to dissect the crosstalk between innate immune signaling and other biological pathways in response to viral infection.

Establishment of Viral Infection and Analysis of Host-Virus Interaction in Drosophila Melanogaster

This protocol describes how to establish viral infection in vivo in Drosophila melanogaster using the nano-injection method and basic techniques to analyze virus-host interaction.

바이러스 성 감염 및 초파리 Melanogaster 상호 작용 호스트-바이러스의 분석의 설립

Virus spreading is a major cause of epidemic diseases. Thus, understanding the interaction between the virus and the host is very important to extend our ...

Methods Article

전신 세균 감염 및 면역 방어 표현형에