The authors thank the Central Animal facility of the University Hospital Aachen for technical support. This work was supported by the German Research Foundation (DFG Ta434/2-1, DFG SFB/TRR 57) and by the Interdisciplinary Center for Clinical Research (IZKF) Aachen. This work was further supported by the Core Facility &#8221;Two-Photon Imaging&#8221;, a Core Facility of the Interdisciplinary Center for Clinical Research (IZKF) Aachen within the Faculty of Medicine at RWTH Aachen University.

참고 : 실험 (NIH 간행물, 8 판, 2011) &#39;관리 및 사용 실험 동물의를위한 안내서&#39;다음 동물 연구에 적용되는 독일 법규에 따라 수행하고, 동물의 보호에 관한 지침 63분의 2,010 / EU 과학적인 목적 (유럽 연합의 공식 저널, 2010)에 사용됩니다. 공식 권한은 정부의 동물 관리 및 사용 사무실 (LANUV 노르 트라 인베스트 팔렌, 레 클링 하우젠, 독일)에서 수여되었다. 주 : 단기 촬상 생략 할 수있는 단계는 제조 시간을 단축하고 수술 프로토콜을 간단하게 별표 (*)로 표시된다 (예를 들면도 촬영, 3-D 스택 또는 짧은 기간 경과 현미경 스냅). 그러나 필요한 생존 시간이 현저히 감소 될 경우 이미징 또한 광범위한 모니터링 및 제어를 순환시키지 않고 수행 될 수있다. 1. 현미경의 설치 및 사전 수술 준비 (5 ~ 10 마일N) <ol><li> (현미경, 전력 연구소, 레이저, 전기 패드, 기후 챔버, 웹 캠, 주사기 펌프 장치, 인공 호흡기)에 스위치 시스템. </li><li> 1.5 부피 %에 O 2 아이소 플루 란 증기를 공급하고 설정 아이소 플루 란 수준을 연결합니다 (V는 / V), 작은 동물 인공 호흡기에 연결합니다. <ol><li> 단기 이미징, 초기 IP 마취 유도 후 이소 플루 란을 이용하여 마취를 유지한다 (단계 2.2 참조). 이어서 안정 간 미세 순환을 보장하는 2 부피 % (v / v)의 아이소 플루 란을 사용하고, 2 시간 미만 촬상 시간을 유지한다. </li></ol></li><li> 150 ~ 200 μL의 볼륨 120-140 호흡 사이클 / 분에 호흡을 설정합니다. </li><li> 마우스 아가로 오스 단계를 설정합니다. 40 °의 각도에서 접시 (약 10cm X 15cm베이스)로 쏟아져, 3 % (w / v) 아가로 오스 100 ㎖를 준비한다. </li><li> 필요한 장비를 수집하는 수술 작업 영역을 설정합니다. 미생물 감염을 방지하기 위해, Sekusept 포르테 S의 1 % 용액 (사용 악기를 9.9 % / V w 소독), Glutaral V / w 9.8 %, 실험 (그림 1A 전에 5 분 동안 포름 알데히드). </li><li> 나머지 구성 요소를 얻 피부 소독제 (예를 들면, 10 %, 포비돈 요오드 용액), 간 스테이지 (스택 및 다리), 아가로 오스 용액 (3 % (w / v)의 PBS)에서 5 %, 주사기 펌프 (W / V) 포도당 용액을 마취 용액 I (케타민 0.1 ㎎ / ㎖, 크 실라 0.5 ㎎ / ㎖, 펜타닐 5 ㎍ / ㎖), 면봉, N- 부틸 -2- Cyanoacrylat, 1X PBS와 함께 (G-5), 시린지 펌프. 예열 배양 챔버에 아가로 오스 단계 접시를 넣어. </li></ol> 2. 기관 절개술 (10 ~ 15 분) <ol><li> 25-28g 무게 하우스 사육에서 C57BL / 6 마​​우스를 사용합니다. 하우스 12 시간 조명 / 12 시간 어두운주기와 온도 -에서 FELASA의 지침에 따라, 습도 제어 환경에서 무균 조건 하에서 마우스. 동물에게 표준 마우스 다이어트 (냄새를 맡아 표준 설치류 다이어트)와 물을 자유 섭취에 무료로 액세스 할 수 있습니다. </li><li> 초기화하여 마우스를 마취원점이라 복강 내 마취 솔루션 II 250 μL (케타민 0.1 ㎎ / ㎖, 크 실라 1 ㎎ / ㎖, 부 프레 놀핀 10 ㎍ / ml)에의 (IP) 주입. <ol><li> * 생체 내에 촬상 동안 유지비를 들어, 연속적으로 연속 IP 주입하여 마취 용액 I를 관리 (케타민 0.1 ㎎ / ㎖, 크 실라 0.5 ㎎ / ㎖, 펜타닐 5 ㎍ / mL)을 0.2 ㎖ / hr의 유량으로 주사기 펌프 장치를 이용 inhalative 이소 플루 란 0.5 부피 % 조합 (v / v)로. </li><li> , 기관 절개술을 위해 피부를 소독, 5 분 (집게로 예를 들어 핀치 발 패드) 후 반사를 확인 마우스가 수술 허용 마취 상태에있는 경우 준비를 시작합니다. </li><li> (그림 1B)를 건조로부터 각막을 방지하기 위해 눈 보호 크림 (예를 들어, Dexpanthenol 50 ㎎ / g)을 적용합니다. </li></ol></li><li> 사지에 대한 접착 테이프를 사용하여 복부 측면을 노출 준비 테이블에 마우스를 흥분시키는; 부드럽게,이 위치에 고정하세요 예. 목을 잡아 늘, 고무 밴드를 사용하여 t을 푹앞니 오. <ol><li> 면봉으로 소독을 적용하여, 포비돈 요오드 용액을 이용하여 피부와 모피를 소독. </li></ol></li><li> 직접 턱 아래에 초기 피부 인하 (0.5 cm 길이)을 수행 (그림 1C) <ol><li> 조심스럽게 침샘 (그림 1D) 사이의 결합 조직을 해부하다. </li><li> 조심스럽게 개방 근육 튜브 주위 기관 (그림 1E, F)를 눈물. </li></ol></li><li> 나중에 환기 튜브 고정 (그림 1G)에 대한 기관 아래 수술 스레드를 놓습니다. <ol><li> T 자형 절개를 수행 연골 고리 사이에 마이크로 가위로 열기 기관 (그림 1H) </li></ol></li><li> 절개 (~ 0.5 cm)를 통해 기관에 환기 튜브를 놓습니다. 앞으로 튜브를 밀어 작은 해부 집게로 꼬리 끝을 잡고 부드럽게 기관지에 튜브를 사전에 (그림 1I) </li><li> 수술 스레드와 튜브를 흥분시키는 (예 : </e스레드 m&gt;, 5-0) 단계 2.5 기관 내부 튜브 주위에 매듭을 묶는에 배치; 실수로 depositioning (그림 1J, K)을 방지하기 위해 피부에 튜브의 두 번째 두개골 고정을 수행합니다. </li><li> 조직 접착제 (예를 들면, n- 부틸 -2- Cyanoacrylat) (그림 1L)와 인감 잘라. </li><li> 접착 테이프를 사용하여 머리에 튜브를 흥분시키는. </li></ol> 3. Laparatomy (15 ~ 20 분) <ol><li> 저체온증을 방지하기 위해 가열 패드에 마우스를 놓습니다. 복부를 면도 조심스럽게 피부에서 머리를 제거합니다. 포비돈 요오드 용액을 사용하여 면도 모피 소독. </li><li> 수술 가위 (그림 2A)를 사용하여 흉골 아래로 잘라 작은 피부를 수행합니다. 출혈을 방지하기 위해 모든 눈에 보이는 혈관을 소작, 측면 양쪽에 갈비뼈 아래 컷을 확장합니다. </li><li> 조심스럽게 복막 (그림 2B)를 열고, 흉골 아래의 원격 교육 알바에 작은 상처를 수행합니다. cauteriz를 사용하여 양쪽에 상처를 확장ATION (그림 2C, D)를 출혈 방지합니다. </li><li> 아가로 오스 단계 접시 (그림 2E)에 마우스를 놓습니다. 마우스 (일반적으로 12 ~ 14 커버 슬립) (그림 2 층)의 양쪽에 적절한 높이의 스택을 놓습니다. </li><li> 호흡과 현미경을 동기화하는 등 위쪽 아래 호흡 트리거 센서를 놓습니다. 트리거 장치 (그림 2G)를 활성화합니다. </li><li> 리브 (그림 2I)을 철회 흉골을 통해 수술 스레드 (5-0) 놓습니다. </li><li> 조심스럽게 곡선 수술 가위를 사용하여 간, 다이어프램 (낫 모양의 인대)를 연결하는 인대뿐만 아니라 간, 위장관를 잘라. 대동맥 (그림 2J)까지 낫 모양의 인대를 잘라. </li></ol> 4. 샘플 설정 (10 ~ 15 분) <ol><li> 큰 간 엽에 쉽게 액세스 할 수 있도록 45 °의 각도로 오른쪽 마우스를 놓습니다. </li><li> 복부를 덮고, 갈비뼈 아래 준비 브리지 추가공동. 예리한 에지 (도 2K)을 덮도록 접착 테이프 코팅 표준 커버 슬립을 사용하여 다리 제조. </li><li> 무대에서 큰 간 로브를 놓습니다. 조심스럽게 이중 볼 스타일러스 프로브 또는 간 아래 패딩 주걱을 미끄러 젖은 면봉이나 물티슈를 사용하여 기관의 상단을 누르고 있습니다. 슬라이드에 로브를 들어 올리고 부드러운 드래그을 적용합니다. 로브가 휘거나 접을 수 있습니다. 간 (그림 2L) 만 부드러운 조작에 각별한주의를 제공합니다. </li><li> 예를 들어, 작은 커버 슬립 (20mm X 20mm), 옆에있는 간 엽의 거의 동일한 높이의 더미 커버 슬립을 지원하기 위해 측면 지원 지분을 놓습니다. </li><li> 간 엽에 큰 커버 슬립 (24mm × 50 mm)를 놓습니다. 그 커버 슬립을 확인하는 것은 가능합니다 (그림 2M)으로 수평으로 배향된다. 주 : 커버 슬립을 압박하지 않고 조직과 접촉해야한다. 장애인 혈액의 흐름 (흰색 조직)의 흔적이 없는지 확인합니다. microcirc 경우위험률이 중단되고, 추가로 지분을 지원하고 추가 할 수 있습니다. </li><li> * 장기 마취 및 G-5 응용 프로그램에 대 한 장소 두 개의 독립적 인 복강 내 카테터 (그림 2N). 다음 뒷다리로 하복부에 횡 방향으로 카테터를 설치합니다. 참고 : 복강 내 카테터는 자체 제작 유연한 실리콘 튜브 (그림 3)과 27 G 바늘을 연결하여 할 수 있습니다. </li><li> * 피부에 5-0 수술 스레드의 루프를 흥분시키는 바늘 실수 변위를 방지 할 수 있습니다. </li><li> * I 1 카테터 마취 용액과 주사기 펌프를 장착, 0.2 ㎖ / 시간으로 설정 유량; 0.1 ㎖ / 시간에 카테터 2 G-5 주사기 펌프, 설정 유량을 연결합니다. </li></ol> 5. 마우스 모니터링 <ol><li> * 장소 ECG 전면에 전극과 뒷다리 사지 (그림 4A). </li><li> * 2 레벨 센서를 공동 만료 튜브를 연결합니다. </li><li> (그림 4C 열 패드에 연결된 외부 온도 센서를 추가). </li></ol> 6. 임베딩 및 조직 고정 (5 ~ 10 분) <ol><li> 3 % 1X PBS에서 (w / v) 아가로 오스 100 ㎖를 준비합니다. 온도가 5 ML의 주사기와 18 G 바늘 (그림 5A)를 사용하여 41 ° C 일 때 간을 포함합니다. </li><li> 커버 슬립에 마우스 (그림 5B, C)의 주위에 남아있는 아가로 오스를 붓고. 아가로 오스가 완전히 호화 때까지 기다립니다. </li><li> 준비 간 엽 (그림 5D, E)를 스캔하기에 충분한 viewfield 큰 준비, 하이 데르 주걱을 사용하여 초과 아가로 오스를 제거합니다. </li></ol> 7. 영상 <ol><li> 현미경 기후 챔버에 마우스를 전송합니다. </li><li> 1X PBS의 50 ~ 100 ML을 추가 (37 ° C에 예열). </li><li> 증발 (그림 5 층)을 방지하기 위해 샘플 접시를 커버. </li><li> * 0.5 권 %로 inhalative 이소 플루 란을 감소 (v / v)로. </li><li> *, ECG 모니터링 소프트웨어, 심박수 및 호기 CO 2 녹화를 시작. </li><li> 영상을 시작합니다 라 열산이 셔터는 관심보기 필드를 식별 Z-스택에 대한 상부 및 하부 경계를 정의하고, 시간 경과 녹화를 시작합니다. Z-드리프트를 해결하기 위해 필요한 경우 (Z-스택을 다시 조정 예. 때문에 혈액의 압력 또는 온도 변화, 그림 (5 세대)의 변화. 주 : 동물의 순환 또는 마취 영상주기의 경우 또는 완성이 불안정하게되면 (마취로부터 각성)없이 경추 탈구하여 쥐를 희생. </li></ol>

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The authors disclose no con&#64258;ict of interests.

본 연구의 목적은 간 생체 내에 TPLSM 이미징 높은 표준화 안정적이고 재현성있는 방법을 개발하는 것이었다. 일반적으로 생체 내에 이미징은 원점 복귀 및 개발, 항상성과 질병에 다른 백혈구 인구의 상호 작용 다음과 같은 실제 생활 조건에서 세포의 행동에 대한 가치있는 통찰력을 부여하고있다. 그러나, 직접적으로 고형 장기에 비해 간뿐만 아니라 높은 취약성으로 인해 전송되는 호흡기 및 장...

세포 및 전체 기관의 세포 기능 또는 전체 유기체의 시각화는 본체 (2)의 거의 모든 부분을 포함, 50 년 이상 큰 관심이었다. 따라서 일부 초기 연구는 이미 간 3,4의 생체 내에 이미지를 고용했다. 그러나, 다음과 같은 몇 가지 제한 사항이 간 조직의 장기 안정적인 고해상도 영상에 관한 최신 상태로 존재한다. 다이어프램 인해 위장관 5 밀착 간 해부학 위치로, 미세한 생체 내에 이미징을위한 가장 일반적인 문제는 장관 (6)의 낮은 정도, 연동 운동 호흡 때문이며. 고형 장기에 비해, 간 수술 특히 도전적이다. 때문에 조밀 한 미세 혈관 구조에, 수술 조작, 대규모 출혈성 병변 거주자의 장애 미세 7도 활성화를 이끌 수 있습니까이러한 쿠퍼 세포 (8)과 같은 mmune 세포. 따라서, 조직의 기계적인 고정화 -6,9-가 생체 내에 현미경 이미징에 간섭 가능성이 다른 공표. 건강한 간에서, 총 혈액량 10~15 %가 간 혈관 내에 존재하고, 기관은 순환의 변화 (예를 들면, 혈압 변동에 매우 민감 장기 렌더링 전체 심 박출량 (10)의 약 25 %를 ). 따라서, 과도한 조직 처리 또는 중앙 집중식 순환에 의해 예를 들면, 전단 응력, 변위, 부상으로 간 혈액 흐름의 중단뿐만 아니라 간 면역 반응에 영향을 미치는, 백혈구 철새 행동의 인공 변경, 손상된 간 산소 때문에 더 간 손상으로 이어질 것 장기 보존과 동물의 전체 수명 등. 초기 현미경 연구는 생체 내에 표면 형광 마일 기반으로했다croscopy 있으나, 이러한 포토 블리치 낮은 침투 깊이와 같은 여러 기술적 인 제약은 장기 간 촬상 4,11,12 본 기술의 사용을 제한한다. 이 새로운 방법은 실생활 13-15에서 거의 모든 장기에 영상 연구를 수행 할 기술적으로 가능했다으로 1990 년대에 다 광자 현미경의 발달과 함께, 사진 표백 또는 침투 깊이의 한계는 주로 해결되었다. 그러나, 간 영상에 대하여 주 남은 과제였다 : 몇 시간 이상 16 시간 동안 사인 곡선의 불변 간 혈류, 특히 안정된 촬상 확보 호흡 운동, 간 조직의자가 형광이. 여러 연구는 간에서 17 예, NKT 세포 18-20, 21, 22 T 세포, 대 식세포 간 23,24 또는 25 호중구, 장기 광자 m 함수를 다양한 백혈구의 이동을 해결되지만icroscopy 영상은 아직 성공적으로 인해 추가 손상 (26)에 대한 기존의 손상 때문에 더 높은 감수성에 작업이 훨씬 더 도전 급성 또는 만성 간 질환 동물에서 설립되지 않았다. 그러나, 실시간으로 간에서 백혈구의 철새 행동과 세포 기능을 모니터링 간 항상성과 질병 (27)에서의 특정 역할에 새로운 통찰력을 할 수 있습니다. 케모카인 수용체 CXCR6 자연 킬러 (NK) 세포, NKT 세포 및 일부 T 세포를 포함한 여러 18,28 개체군 림프구 서브 세트 상에 발현된다. 마우스에서의 연구는 이전 CXCR6와 동족 리간드 CXCL16 항상성 동안 간 정​​현파에 NKT 세포의 순찰을 제어 할 수 있음을 나타내었다. 결과적으로, (CXCR6 궤적에 녹색 형광 단백질 [GFP]의 노크에 들고) CXCR6.gfp 마우스의 사용은 뇌 29 등의 각종 장기에 림프구의 이동을 조사 설명 하였지만또한 간 (18, 20)는, 염증에 CXCR6.gfp 세포의 침윤을 증가 게재. 본 연구에서 제공된 방법에 안정화 된 조건 하에서 장기간에 걸쳐 이러한 프로세스를 수행 할 수 있었다. 생체 내에 광자 기반의 절차는 동물과 기관의 최소한의 교란과 높은 재현성했다 이미징을 허용; 호흡과 순환의 가까운 제어 다음에 광범위한 모니터링하여 장기 동물의 생존을 위해 최적화; 매우 유연하고 쉽게는 신장이나 비장 등의 실질 장기로도 채택한다.

<table border="0" cellpadding="0" cellspacing="0" width="1184">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Anesthetics</td>
			<td style="width:263px;"></td>
			<td style="width:219px;"></td>
			<td style="width:293px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Buprenorphine</td>
			<td style="width:263px;">Essex Pharma</td>
			<td style="width:219px;">997.00.00</td>
			<td>Analgeticum, 0.1 mg/kg</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Fentanyl</td>
			<td style="width:263px;">Rotex Medica</td>
			<td style="width:219px;">charge: 30819</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Fluovac anesthesia system</td>
			<td style="width:263px;">Harvard Apparatus</td>
			<td style="width:219px;">34-1030</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Glucose 5%</td>
			<td style="width:263px;">Braun</td>
			<td style="width:219px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ISOFLO (Isoflurane Vapor) vaporiser</td>
			<td style="width:263px;">Eickemeyer</td>
			<td style="width:219px;">4802885</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Isoflurane</td>
			<td style="width:263px;">Forene Abbott</td>
			<td style="width:219px;">B 506</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Isotonic (0.9%) NaCl solution</td>
			<td style="width:263px;">DeltaSelect GmbH</td>
			<td style="width:219px;">PZN 00765145</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Ketamin 10%</td>
			<td style="width:263px;">ceva</td>
			<td style="width:219px;">Charge: 36217/09</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;"></td>
			<td style="width:263px;"></td>
			<td style="width:219px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Xylazin 2%</td>
			<td style="width:263px;">medistar</td>
			<td style="width:219px;">Charge: 04-03-9338/23</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;"></td>
			<td style="width:263px;"></td>
			<td style="width:219px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Consumable supplies</td>
			<td style="width:263px;"></td>
			<td style="width:219px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">20ml Syringe</td>
			<td style="width:263px;">BD Plastipak</td>
			<td style="width:219px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">250ml Erlenmeyer flask</td>
			<td style="width:263px;">Schott Duran</td>
			<td>21 226 36</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">25mL Beaker 2x</td>
			<td style="width:263px;">Schott Duran</td>
			<td style="width:219px;">50-1150</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">2ml syringe</td>
			<td style="width:263px;">BD Plastipak</td>
			<td style="width:219px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">4-0 Vicryl suture</td>
			<td style="width:263px;">Ethicon</td>
			<td style="width:219px;">V7980</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Agarose</td>
			<td style="width:263px;">commercially available</td>
			<td style="width:219px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Bepanthen Eye and Nose ointment</td>
			<td style="width:263px;">Bayer Vital GmbH</td>
			<td style="width:219px;">6029009.00.00</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Change-A-Tip Deluxe High-Temp Cautery Kit</td>
			<td style="width:263px;">Fine Science Tools Inc.</td>
			<td style="width:219px;">18010-00</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Cotton Gauze swabs</td>
			<td style="width:263px;">Fuhrmann GmbH</td>
			<td style="width:219px;">32014</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Cover Slip 24x50mm</td>
			<td style="width:263px;">ROTH</td>
			<td style="width:219px;">1871</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Durapore silk tape</td>
			<td style="width:263px;">3M</td>
			<td style="width:219px;">1538-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Feather disposable scalpel</td>
			<td style="width:263px;">Feather</td>
			<td style="width:219px;">02.001.30.011</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Fine Bore Polythene Tubing 0,58mm ID</td>
			<td style="width:263px;">Smiths medical</td>
			<td style="width:219px;">800/100/200</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Histoacryl</td>
			<td style="width:263px;">Braun</td>
			<td style="width:219px;">1050052</td>
			<td>5x 0,5ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Leukoplast</td>
			<td style="width:263px;">BSN Medical Inc.</td>
			<td style="width:219px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Microscope Slides</td>
			<td style="width:263px;">ROTH</td>
			<td style="width:219px;">1879</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Poly-Alcohol Haut&#8230;farblos Antisepticum</td>
			<td style="width:263px;">Antiseptica GmbH</td>
			<td style="width:219px;">72PAH200</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Sterican needle 18 G x 1</td>
			<td style="width:263px;">B. Braun</td>
			<td style="width:219px;">304622</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Sterican needle 27 3/4 G x 1</td>
			<td style="width:263px;">B. Braun</td>
			<td style="width:219px;">4657705</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Tissue paper</td>
			<td style="width:263px;">commercially available</td>
			<td style="width:219px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;"></td>
			<td style="width:263px;"></td>
			<td style="width:219px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Surgical Instruments</td>
			<td style="width:263px;"></td>
			<td style="width:219px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Amalgam burnisher 3PL</td>
			<td style="width:263px;">Gatz</td>
			<td style="width:219px;">0110?</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Blair retractors (4 pronged (blunt)) x2</td>
			<td style="width:263px;">Storz&#38;Klein</td>
			<td style="width:219px;">S-01134</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dumont No.7 forceps</td>
			<td style="width:263px;">Fine Science Tools Inc.</td>
			<td style="width:219px;">91197-00</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Graefe forceps curved x1</td>
			<td style="width:263px;">Fine Science Tools Inc.</td>
			<td style="width:219px;">11151-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Graefe forceps straight x2</td>
			<td style="width:263px;">Fine Science Tools Inc.</td>
			<td style="width:219px;">11050-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Heidemann spatula HD2</td>
			<td style="width:263px;">Stoma</td>
			<td style="width:219px;">2030.00</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Needle holder Mathieu</td>
			<td style="width:263px;">Fine Science Tools Inc.</td>
			<td style="width:219px;">12010-14</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Scissor</td>
			<td style="width:263px;">Fine Science Tools Inc.</td>
			<td style="width:219px;">14074-11</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Semken forceps</td>
			<td style="width:263px;">Fine Science Tools Inc.</td>
			<td style="width:219px;">11008-13</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Small surgical scissors curved</td>
			<td style="width:263px;">Fine Science Tools Inc.</td>
			<td style="width:219px;">14029-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Small surgical scissors straight</td>
			<td style="width:263px;">Fine Science Tools Inc.</td>
			<td style="width:219px;">14028-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Standard pattern forceps</td>
			<td style="width:263px;">Fine Science Tools Inc.</td>
			<td style="width:219px;">11000-12</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vannas spring scissors</td>
			<td style="width:263px;">Fine Science Tools Inc.</td>
			<td style="width:219px;">15000-08</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;"></td>
			<td style="width:263px;"></td>
			<td style="width:219px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Equipment</td>
			<td style="width:263px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">ECG Trigger Unit</td>
			<td style="width:263px;">Rapid Biomedical</td>
			<td style="width:219px;">3000003686</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">MICROCAPSTAR End-Tidal Carbon Dioxide Analyzer</td>
			<td style="width:263px;">AD Instruments</td>
			<td style="width:219px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Minivent Typ 845</td>
			<td style="width:263px;">Harvard Apparatus</td>
			<td>73-0043</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Multiphoton microscope Trimscope I</td>
			<td style="width:263px;">LaVision</td>
			<td style="width:219px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Perfusor Compact</td>
			<td style="width:263px;">B. Braun</td>
			<td style="width:219px;"></td>
			<td></td>
		</tr>
		<tr height="21">
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long term intravital multiphoton microscopy imaging of immune cells in healthy and diseased liver using cxcr6.gfp reporter mice

<html> 부상에 대한 응답으로서 간염은 단핵구, 호중구, T 세포의 서브 세트, B 세포, 자연 살해 (NK) 및 NKT 세포의 구별을 포함한 백혈구 아형의 침입을 수반하는 매우 동적 인 과정이다. 면역 세포의 이동을 모니터링하기위한 생체 내에 간 현미경 인해 시료 전처리 및 고정, 광학 해상도 및 장기 생존 동물에 대한 높은 요구에 특히 도전적이다. 그러나, 염증 과정뿐만 아니라, 셀룰러 상호 작용 연구 역학 잘 염증성 간 질환의 개시, 진행 및 회귀 이해에 중요한 정보를 제공 할 수있다. 따라서, 매우 민감하고 신뢰할 수있는 방법은 집중 치료와 함께 마이그레이션 및 생체 내에 두 광자 레이저 스캐닝 현미경 (TPLSM)에 의해 오랜 기간 (약 6 시간)을 마우스 간에서 다른 면역 세포의 세포 - 세포 상호 작용을 연구하기 위해 설립되었습니다 모니터링. 이비인후과 &quot;&gt; 제공 방법은 부드러운 준비 및 기관의 최소 섭동과 간의 안정적인 고정을 포함하며, 장기간 생체 내에 촬상 추적을 허용하는, 최대 6 시간의 기간 동안 사실상 광표백 또는 광독성 효과 다색 다 광자 현미경을 사용하여 마우스 중요한 매개 변수와 순환, 온도, 가스 교환의 안정화의 광범위한 모니터링에 의한 안정적인 영상 조건, 특정 백혈구 하위 집합. CXCR6.gfp 간염시 림프구 마이그레이션을 조사하기 위해 넉 - 생쥐 기준선 조건 하에서 사염화탄소 복강 내 주사 발 CCL (4)에 의해 유도되는 급성 및 만성 간 손상 후 생체 내에 간 촬상을 실시한다. 천연 킬러 (NK) - - 또한 그러한 CD4 T 세포로서 T 림프구 서브 세트했지만 점막 associ CXCR6 주로 자연 살해 T (NKT)에 림프구상에서 발현 케모카인 수용체이며ated의 불변 (MAIT) T 세포 하나. 간 손상에 따라 자신의 변화된 행동에 대한 상세한 통찰력과 질병의 진행에 따라서 잠재적 인 참여를 허용 CXCR6.gfp + 면역 세포의 이주 패턴과 위치에 따라.

우리의 생체 내에 TPLSM 접근 방식의 유효성을 확인하기 위해, 우리는 생체 내에 TPLSM 이미징에 CXCR6의 GFP / + 마우스를 실시. 마우스를 어느 기준선 대조군으로 방치 또는 급성 간 손상 유도 20 사염화탄소 단일 복강 내 주사 (사염화탄소)을 실시 하였다. 녹색 형광 인해 비디오 시퀀스는 2-5 시간의 기간 동안 수행 한 후, 세포는 시간이 지남에 따라 추적 ?...

Watch this Scientific Journal Video about Long Term Intravital Multiphoton Microscopy Imaging of Immune Cells in Healthy and Diseased Liver Using CXCR6.Gfp Reporter Mice at JoVE.com

Long Term Intravital Multiphoton Microscopy Imaging of Immune Cells in Healthy and Diseased Liver Using CXCR6.Gfp Reporter Mice

Stable intravital high-resolution imaging of immune cells in the liver is challenging. Here we provide a highly sensitive and reliable method to study migration and cell-cell-interactions of immune cells in mouse liver over long periods (about 6 hours) by intravital multiphoton laser scanning microscopy in combination with intensive care monitoring.

CXCR6.Gfp 기자 마우스를 사용하여 건강하고 병에 걸린 간에서 면역 세포의 장기 생체 내에 다중 광자 현미경 이미징

Liver inflammation as a response to injury is a highly dynamic process involving the infiltration of distinct subtypes of leukocytes including monocytes, ...

long-term-intravital-multiphoton-microscopy-imaging-immune-cells

Research

JoVE Journal

Immunology and Infection

11.8K 조회수.  RWTH University-Hospital Aachen. Stable intravital high-resolution imaging of immune cells in the liver is challenging. Here we provide a highly sensitive and reliable method to study migration and cell-cell-interactions of immune cells in mouse liver over long periods (about 6 hours) by intravital multiphoton laser scanning microscopy in combination with intensive care monitoring.

비디오: Long Term Intravital Multiphoton Microscopy Imaging of Immune Cells in Healthy and Diseased Liver Using CXCR6.Gfp Reporter Mice

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

Intravital Imaging of the Mouse Thymus using 2-Photon Microscopy

Two-photon Microscopy (TPM) provides image acquisition in deep areas inside tissues and organs. In combination with the development of new stereotactic tools and surgical procedures, TPM becomes a powerful technique to identify "niches" inside organs and to document cellular "behaviors" in live animals. While intravital imaging provides information that best resembles the real cellular behavior inside the organ, it is both more laborious and technically demanding in terms of required equipment/procedures than alternative ex vivo imaging acquisition. Thus, we describe a surgical procedure and novel "stereotactic" organ holder that allows us to follow the movements of Foxp3+ cells within the thymus.
Foxp3 is the master regulator for the generation of regulatory T cells (Tregs). Moreover, these cells can be classified according to their origin: ie. thymus-differentiated Tregs are called "naturally-occurring Tregs" (nTregs), as opposed to peripherally-converted Tregs (pTregs). Although significant amount of research has been reported in the literature concerning the phenotype and physiology of these T cells, very little is known about their in vivo interactions with other cells. This deficiency may be due to the absence of techniques that would permit such observations. The protocol described in this paper provides a remedy for this situation.
Our protocol consists of using nude mice that lack an endogenous thymus since they have a punctual mutation in the DNA sequence that compromises the differentiation of some epithelial cells, including thymic epithelial cells. Nude mice were gamma-irradiated and reconstituted with bone marrows (BM) from Foxp3-KIgfp/gfp mice. After BM recovery (6 weeks), each animal received embryonic thymus transplantation inside the kidney capsule. After thymus acceptance (6 weeks), the animals were anesthetized; the kidney containing the transplanted thymus was exposed, fixed in our organ holder, and kept under physiological conditions for in vivo imaging by TPM. We have been using this approach to study the influence of drugs in the generation of regulatory T cells.

We have developed novel laboratory tools and protocols for intravital imaging acquisition of the thymus. Our technique should help in the identification of &#x201C;niches&#x201D; within the thymus where T cell development occurs.

2 - 광자 현미경을 사용하여 마우스 Thymus의 Intravital 영상

Two-photon Microscopy (TPM) provides image acquisition in deep areas inside tissues and organs. In combination with the development of new stereotactic ...

Long Term Chronic Pseudomonas aeruginosa Airway Infection in Mice

A mouse model of chronic airway infection is a key asset in cystic fibrosis (CF) research, although there are a number of concerns regarding the model itself. Early phases of inflammation and infection have been widely studied by using the Pseudomonas aeruginosa agar-beads mouse model, while only few reports have focused on the long-term chronic infection in vivo. The main challenge for long term chronic infection remains the low bacterial burden by P. aeruginosa and the low percentage of infected mice weeks after challenge, indicating that bacterial cells are progressively cleared by the host.
This paper presents a method for obtaining efficient long-term chronic infection in mice. This method is based on the embedding of the P. aeruginosa clinical strains in the agar-beads in vitro, followed by intratracheal instillation in C57Bl/6NCrl mice. Bilateral lung infection is associated with several measurable read-outs including weight loss, mortality, chronic infection, and inflammatory response. The P. aeruginosa RP73 clinical strain was preferred over the PAO1 reference laboratory strain since it resulted in a comparatively lower mortality, more severe lesions, and higher chronic infection. P. aeruginosa colonization may persist in the lung for over three months. Murine lung pathology resembles that of CF patients with advanced chronic pulmonary disease.
This murine model most closely mimics the course of the human disease and can be used both for studies on the pathogenesis and for the evaluation of novel therapies.

Long Term Chronic Pseudomonas aeruginosa Airway Infection in Mice

We describe the agar-beads method to establish persistent long-term chronic Pseudomonas aeruginosa airway infection in the mouse model.

장기 만성<em&gt; 녹농균</em마우스의&gt;기도 감염

A mouse model of chronic airway infection is a key asset in cystic fibrosis (CF) research, although there are a number of concerns regarding the model ...

Highly Resolved Intravital Striped-illumination Microscopy of Germinal Centers

Monitoring cellular communication by intravital deep-tissue multi-photon microscopy is the key for understanding the fate of immune cells within thick tissue samples and organs in health and disease. By controlling the scanning pattern in multi-photon microscopy and applying appropriate numerical algorithms, we developed a striped-illumination approach, which enabled us to achieve 3-fold better axial resolution and improved signal-to-noise ratio, i.e. contrast, in more than 100 &#181;m tissue depth within highly scattering tissue of lymphoid organs as compared to standard multi-photon microscopy. The acquisition speed as well as photobleaching and photodamage effects were similar to standard photo-multiplier-based technique, whereas the imaging depth was slightly lower due to the use of field detectors. By using the striped-illumination approach, we are able to observe the dynamics of immune complex deposits on secondary follicular dendritic cells &#8211; on the level of a few protein molecules in germinal centers.

High-resolution intravital imaging with enhanced contrast up to 120 &#181;m depth in lymph nodes of adult mice is achieved by spatially modulating the excitation pattern of a multi-focal two-photon microscope. In 100 &#181;m depth we measured resolutions of 487 nm (lateral) and 551 nm (axial), thus circumventing scattering and diffraction limits.

어린 싹 센터의 높은 해결의 intravital 스트라이프 - 조명 현미경

Monitoring cellular communication by intravital deep-tissue multi-photon microscopy is the key for understanding the fate of immune cells within thick ...

Intravital Microscopy Imaging of the Liver following Leishmania Infection: An Assessment of Hepatic Hemodynamics

Intravital microscopy (IVM) is a powerful optical imaging technique that has made possible the visualization, monitoring and quantification of various biological events in real time and in live animals. This technology has greatly advanced our understanding of physiological processes and pathogen-mediated phenomena in specific organs.
In this study, IVM is applied to the mouse liver and protocols are designed to image in vivo the circulatory system of the liver and measure red blood cell (RBC) velocity in individual hepatic vessels. To visualize the different vessel subtypes that characterize the hepatic organ and perform blood flow speed measurements, C57Bl/6 mice are intravenously injected with a fluorescent plasma reagent that labels the liver-associated vasculature. IVM enables in vivo, real time, measurement of RBC velocity in a specific vessel of interest. Establishing this methodology will make it possible to investigate liver hemodynamics under physiological and pathological conditions. Ultimately, this imaging-based methodology will be important for studying the influence of L. donovani infection&#160;on hepatic hemodynamics.
This method can be applied to other infectious models and mouse organs and might be further extended to pre-clinical testing of a drug&#8217;s effect on inflammation by quantifying its effect on blood flow.

Intravital Microscopy Imaging of the Liver following Leishmania Infection: An Assessment of Hepatic Hemodynamics

This article reports on a detailed method for the dynamic measurement and quantification of blood flow velocity within individual blood vessels of the mouse liver vasculature using intravital microscopy imaging in combination with a specific methodology for image acquisition and analysis.

다음 간 생체 내에 현미경 이미징<em&gt; 슈만 편모충</em&gt; 감염 : 간 혈류의 평가

Intravital microscopy (IVM) is a powerful optical imaging technique that has made possible the visualization, monitoring and quantification of various ...

Intravital Microscopy of Leukocyte-endothelial and Platelet-leukocyte Interactions in Mesenterial Veins in Mice

Intravital microscopy is a method that can be used to investigate different processes in different regions and vessels in living animals. In this protocol, we describe intravital microscopy of mesentery veins. This can be performed in a short period of time with reproducible results showing leukocyte-endothelial interactions in vivo. We describe an inflammatory setting after LPS challenge of the endothelium. But in this model one can apply many different types of inflammatory conditions, like bacterial, chemical or biological and investigate the administration of drugs and their direct effects on the living animal and its impact on leukocyte recruitment. This protocol has been applied successfully to a number of different treatments of mice and their effects on inflammatory response in vessels. Herein, we describe the visualization of leukocytes and platelets by fluorescently labeling these with rhodamine 6G. Additionally, any specific imaging can be performed using targeted fluorescently labeled molecules.

This simplified application of intravital microscopy of mesentery veins in mice can be used in different models of inflammation to observe leukocyte-endothelial and platelet-leukocyte interactions.

마우스의 Mesenterial 혈관에서의 생체 내에 현미경 백혈구 - 내피 세포와 혈소판 - 백혈구 상호 작용

Intravital microscopy is a method that can be used to investigate different processes in different regions and vessels in living animals. In this ...

Analysis of Yersinia enterocolitica Effector Translocation into Host Cells Using Beta-lactamase Effector Fusions

Many gram-negative bacteria including pathogenic Yersinia spp. employ type III secretion systems to translocate effector proteins into eukaryotic target cells. Inside the host cell the effector proteins manipulate cellular functions to the benefit of the bacteria. To better understand the control of type III secretion during host cell interaction, sensitive and accurate assays to measure translocation are required. We here describe the application of an assay based on the fusion of a Yersinia enterocolitica effector protein fragment (Yersinia outer protein; YopE) with TEM-1 beta-lactamase for quantitative analysis of translocation. The assay relies on cleavage of a cell permeant FRET dye (CCF4/AM) by translocated beta-lactamase fusion. After cleavage of the cephalosporin core of CCF4 by the beta-lactamase, FRET from coumarin to fluorescein is disrupted and excitation of the coumarin moiety leads to blue fluorescence emission. Different applications of this method have been described in the literature highlighting its versatility. The method allows for analysis of translocation in vitro and also in in vivo, e.g., in a mouse model. Detection of the fluorescence signals can be performed using plate readers, FACS analysis or fluorescence microscopy. In the setup described here, in vitro translocation of effector fusions into HeLa cells by different Yersinia mutants is monitored by laser scanning microscopy. Recording intracellular conversion of the FRET reporter by the beta-lactamase effector fusion in real-time provides robust quantitative results. We here show exemplary data, demonstrating increased translocation by a Y. enterocolitica YopE mutant compared to the wild type strain.

Analysis of Yersinia enterocolitica Effector Translocation into Host Cells Using Beta-lactamase Effector Fusions

Effector translocation into host cells via a type III secretion system is a common virulence strategy among gram-negative bacteria. A beta-lactamase effector fusion based assay for quantitative analysis of translocation was applied. In Yersinia infected cells, conversion of a FRET reporter by the beta-lactamase is monitored using laser scanning microscopy.

분석<em&gt; 예 르시 니아의 enterocolitica</em숙주 세포 내로&gt; 이펙터 전좌 베타 - 락타 마제 이펙터의 용해 사용

Many gram-negative bacteria including pathogenic Yersinia spp. employ type III secretion systems to translocate effector proteins into eukaryotic target ...

Imaging Neutrophils and Monocytes in Mesenteric Veins by Intravital Microscopy on Anaesthetized Mice in Real Time

Efficient immune response is dependent on rapid mobilization of blood leukocytes to the site of infection or injury. Investigating leukocyte migration in vivo is crucial for understanding the molecular basis of leukocyte transendothelial migration and interaction with vascular endothelium. One powerful approach involves intravital microscopy on transgenic mice expressing fluorescent proteins in cells of interest.
Here we present a protocol for imaging monocytes and neutrophils in the CX3CR1gfp/wt mouse i.v. injected with orange dye-labeled neutrophils with an inverted confocal microscope. Time-lapse movies gathered from 30 min&#160;to several hours of imaging allow the analysis of leukocyte behavior in mesenteric veins under both steady state and inflammatory conditions. We also describe the steps to locally induce blood vessel inflammation with TLR2/TLR1 agonist Pam3SK4 and monitor the subsequent recruitment of neutrophils and monocytes.
The presented technique can also be used to monitor other populations of leukocytes and investigate molecules implicated in leukocyte recruitment or trafficking using other stimuli or transgenic mice.

We detail a protocol to monitor the behavior of neutrophils and monocytes in mesenteric veins under steady state and inflammatory conditions using intravital confocal microscopy on anaesthetized mice.

실시간으로 마취 마우스에 생체 내에 현미경으로 장간막 혈관에서 호중구와 단핵구 이미징

Efficient immune response is dependent on rapid mobilization of blood leukocytes to the site of infection or injury. Investigating leukocyte migration in ...

Intravital Imaging of Neutrophil Priming Using IL-1&#946; Promoter-driven DsRed Reporter Mice

Neutrophils are the most abundant leukocytes in human blood circulation and are quickly recruited to inflammatory sites. Priming is a critical event that enhances the phagocytic functionality of neutrophils. Although extensive studies have unveiled the existence and importance of neutrophil priming during infection and injury, means of visualizing this process in vivo have been unavailable. The protocol provided enables monitoring of the dynamic process of neutrophil priming in living animals by combining three methodologies: 1) DsRed reporter signal &#8212; used as a measure of priming 2) in vivo neutrophil labeling &#8212; achieved by injection of fluorescence-conjugated anti-lymphocyte antigen 6G (Ly6G) monoclonal antibody (mAb) and 3) intravital confocal imaging. Several critical steps are involved in this protocol: oxazolone-induced mouse ear skin inflammation, appropriate sedation of animals, repeated injections of anti-Ly6G mAb, and prevention of focus drift during imaging. Although a few limitations have been observed, such as the limit of continuous imaging time (~ 8 hr) in one mouse and the leakage of fluorescein isothiocyanate-dextran from blood vessels in the inflammatory state, this protocol provides a fundamental framework for intravital imaging of primed neutrophil behavior and function, which can easily be expanded to examination of other immune cells in mouse inflammation models.

Intravital Imaging of Neutrophil Priming Using IL-1&amp;#946; Promoter-driven DsRed Reporter Mice

This current protocol employs fluorescent reporters, in vivo labeling, and intravital imaging techniques to enable monitoring of the dynamic process of neutrophil priming in living animals.

IL-1β의 발기인 기반을 DsRed 기자 마우스를 사용 호중구 초벌의 생체 내에 영상

Neutrophils are the most abundant leukocytes in human blood circulation and are quickly recruited to inflammatory sites. Priming is a critical event that ...

Imaging Mycobacterium tuberculosis in Mice with Reporter Enzyme Fluorescence

Reporter enzyme fluorescence (REF) utilizes substrates that are specific for enzymes present in target organisms of interest for imaging or detection by fluorescence or bioluminescence. We utilize BlaC, an enzyme expressed constitutively by all M. tuberculosis strains. REF allows rapid quantification of bacteria in lungs of infected mice. The same group of mice can be imaged at many time points, greatly reducing costs, enumerating bacteria more quickly, allowing novel observations in host-pathogen interactions, and increasing statistical power, since more animals per group are readily maintained. REF is extremely sensitive due to the catalytic nature of the BlaC enzymatic reporter and specific due to the custom flourescence resonance energy transfer (FRET) or fluorogenic substrates used. REF does not require recombinant strains, ensuring normal host-pathogen interactions. We describe the imaging of M. tuberculosis infection using a FRET substrate with maximal emission at 800 nm. The wavelength of the substrate allows sensitive deep tissue imaging in mammals. We will outline aerosol infection of mice with M. tuberculosis, anesthesia of mice, administration of the REF substrate, and optical imaging. This method has been successfully applied to evaluating host-pathogen interactions and efficacy of antibiotics targeting M. tuberculosis.

Imaging Mycobacterium tuberculosis in Mice with Reporter Enzyme Fluorescence

We describe the optical imaging of mice infected with Mycobacterium tuberculosis (M. tuberculosis) using reporter enzyme fluorescence (REF). This protocol facilitates the sensitive and specific detection of M. tuberculosis in pre-clinical animal models for pathogenesis, therapeutics and vaccine research.

기자 효소 형광와 쥐에서 이미징 결핵균

Reporter enzyme fluorescence (REF) utilizes substrates that are specific for enzymes present in target organisms of interest for imaging or detection by ...