이 작품은 우리가 SLO 발현 플라스미드 오클라호마 대학에서 박사 R. Twete​​n 감사 NWA에 NIH 보조금 R37 AI34867 및 R01 GM064625에 의해 지원되었다.

1. ASM의 전사 침묵 <ol><li> 시드 1.5 X 10 DMEM 성장 매체의 2 ml 중 5 HeLa 세포 35mm 유리 바닥 접시에 (MatTek) (DMEM 높은 10 % 소 태아 혈청 (FBS), 2 ㎜의 L-글루타민, 1 % 펜-Strep의 가진 글루코오스) 및 5 % CO 2 배양기에서 37 ° C에서 밤새 품어. </li><li> 다음 날, 성장 미디어 대기음 및 siRNA의 형질 혼합물을 추가하기 전에 2 ㎖의 DMEM 감소 혈청 배지 (4 % FBS와 DMEM), 최소 1 시간으로 대체. </li><li> siRNA를 올리고 형질 혼합물 4 튜브를 준비합니다. 튜브 및 C에서, 형질 전환되는 35mm 접시 당 250 μL의 티멤 감소 혈청과 4 ㎕의 리포 펙 타민 RNAiMax를 추가합니다. 관 B에서 티멤 250 μL가 형질 전환되는 35mm 접시 당, 제어 매체 GC 콘텐츠 올리고 (컨트롤의 siRNA)의 혈청과 8 μL (160 pmole의)를 감소 추가합니다. 관 D에서 추가 티멤 250 μL는 35mm 접시 t 당 SMPD1 올리고 (siASM)의 혈청과 8 μL (160 pmole의)을 감소오 형질 전환 될 수있다. 5 분간 실온에서 튜브를 배양한다. </li><li> 형질 전환 제어의 siRNA에 대한 복잡하고 ASM의 siRNA에 대한 관 C와 D를 형성하는 관 A와 B를 결합합니다. 20 분 동안 실온에서 반응을 배양한다. </li><li> 각각의 35-mm의 유리 바닥 요리에 컨트롤의 siRNA와 ASM siRNA의 형질 반응 천천히 혼합물, 적하, 추가 37 ° C / 5 % CO 2에서 품어. 24 시간 후 형질 전환에서 부드럽게 미디어를 대기음 신선한 DMEM 성장 용지로 교체합니다. 효율적인 ASM의 최저를 들어, 요리는 더 라이브 영상까지 약 31 시간 (총 55 시간) 동안 37 ° C / 5 % CO 2에서 배양해야한다. </li></ol> 2. 라이브 현미경 용 시약의 조제 <ol><li> 칼슘 2 + 및 Mg를 2 +없이 차가운 1X PBS 100 NG / μL의 농도로 재조합 기공 형성 독소 Streptolysin O (SLO)의 솔루션을 준비합니다. 이러한 분석에 사용 SLO는 constitutivel이며친절 박사로드 Twete​​n, 오클라호마 대학교에서 제공하는 활동에 대한 환원제를 필요로하지 않는 Y 활성 시스테인 돌연변이. 독소는 E. 발현되었다 대장균 BL21 DE3 이전 16 바와 같이, 네이티브 조건에서 정제 하였다. </li><li> FM1-43 원액을 준비 DMSO에 재현 탁 FM1-43 동결 건조 분말은 25 밀리미터 솔루션을 만들 수 있습니다. </li><li> 솔루션을 준비 : 4 μM (1.5 ㎖를 MatTek 요리마다 필요한)의 최종 농도로 칼슘 2 +와 차가운 DMEM에서 FM1-43 원액을 희석하고 필요할 때까지 얼음에 저장합니다. </li><li> 솔루션 B를 준비 4 μM (MatTek 요리에 필요한이 1.5 ㎖)의 최종 농도로 칼슘 2 + 10 mM의 EGTA (칼슘 2 + 무료 DMEM)없이 차가운 DMEM에서 FM1-43 원액을 희석 할 때까지 얼음에 저장 필요. </li><li> 사전 따뜻한 (37 ° C) 에펜 도르프 튜브에 용액 A와 용액 B 1 ㎖ 분량 씩. </li><li> 칼슘 2 +, 그리고 C와 DMEM의 얼음 솔루션을 유지2 +가없는 DMEM. </li></ol> 3. FM1-43의 치료 SLO에 유입 처리하지 않은 세포의 라이브 셀 이미징 <ol><li> , 얼음 조각과 함께 중간 크기 얕은 금속 컨테이너를 채우기 얼음과 큰 유리 용기에 반전, 노출 된 금속 용기 만 바닥면을 떠나 추가로 얼음을 추가합니다. 심지어는 열 전달이 가능하도록 노출 된 금속 바닥에 젖은 종이 타월을 놓습니다. </li><li> 하나의 제어 siRNA를 처리 MatTek 요리 (샘플 1)를 타고 차가운 젖은 종이 타월 위에 놓습니다. 부드럽게 모든 미디어를 대기음 차가운 칼슘 2 + 무료 DMEM으로 배를 세척한다. 마지막 세척 후, 접시에 모든 미디어를 대기음. </li><li> 이미지 세포 관련 FM1-43 없음 SLO의 상흔 칼슘의 존재 (샘플 1)과 세포에서 +, 35-mm의 유리 바닥 접시의 중심 유리 coverslip에 추위 솔루션 B의 180 μl를 추가하고 5 알을 품다 분 얼음에 (표 1). </li&gt; <li> 37 ° C로 가열하고, (온도, 습도, CO 2 수준을 유지) 환경 챔버가 장착 된 공 초점 현미경의 무대에 MatTek 요리를 놓습니다. 40X NA 1.3 목적 (니콘)을보고 이미지를 만들 세포의 필드를 찾을 수 있습니다. 가능한 경우, 온도 드리프트 보정하기 위해 PerfectFocus 또는 이와 유사한 장치를 켭니다. </li><li> 부드럽게 35-mm 접시의 측면에 데워진 솔루션 1 ML을 추가하고 환경 챔버 (표 1)의 커버를 교체합니다. </li><li> (UltraView 복스 퍼킨 엘머 회전 디스크 공 촛점 현미경 시스템에 Volocity) 적절한 이미징 소프트웨어를 사용하여 3 초마다 1 프레임에서 4 분 동안 이미지를 획득. FM1-43의 여자는 488 nm의 밴드 패스와 다이크로 익 필터를 사용하여 488 nm의 레이저 라인으로 수행하고, 배출 차별은 527 (W55) 방출 필터의 사용을 통해 달성된다. 레이저 전력 레벨 및 카메라 민감도는 100 밀리의 화상 노광을 달성하기 위해 설정된다. </li><li> 반복 3.2 3.6에 그 단계 3.5 솔루션 B에 (표 1)를 추가해야합니다 제외하고, 칼슘 2 +의 부재에 SLO의 상흔 (샘플 2)로 제어 siRNA를 처리 한 세포에서 FM1-43를 검출하는 단계를 반복합니다. </li><li> 존재 (샘플 3) 또는 칼슘 2 +의 부재 (샘플 4)의 중심 유리 커버 슬립에 SLO를 포함하는 차가운 솔루션 B의 180 μl를 추가로 하나 SLO로 부상 제어 siRNA를 처리 한 세포로 FM1-43의 유입을 측정하려면 35-mm 유리 바닥 요리와 단계 3.3에서와 같이 얼음에 5 분 동안 품어. 반복 3.6을 통해 3.2 단계, 3.5 단계 (표 1)에서 솔루션 또는 솔루션 B 중 하나를 추가 조정합니다. </li><li> 세포막 수리 ASM의 역할을 확인하려면, 반복 모든 조건 (예제 5-8) (표 1)에 대해 ASM의 siRNA를 처리 한 세포를 사용하여 3.2-3.6 단계를 반복합니다. </li><li> 세포에 재조합 sphingomyelinase (SM)의 역할을 결정하기 위해(FM1-43 유입의 억제에 의해 반사) 세포막 수리의 반응 속도는 (샘플 9 ~ 10), SM은 추가 단계 3.5 (0 ~ 50 μU)에 대한 해결 방법 중 하나 또는 솔루션 B 다음 라이브 세포 중 세포에 추가 1 ml의 (표 1)의 총 부피에서 촬상. </li></ol> 4. 세포에 FM1-43의 유입의 정량화 <ol><li> 영화를 구입 한 후, 이미지 분석 소프트웨어 (Volocity 스위트, 퍼킨 엘머)를 사용하여 FM1-43의 세포 내 형광 강도를 측정한다. 필드에있는 각 셀의 세포질 영역에서 관심 영역을 그리고 비디오의 모든 프레임에 걸쳐 평균 FM1 -​​ 43 형광 강도를 결정하는 소프트웨어 도구를 적용합니다. </li><li> 초기 FM1 -​​ 43 염색의 변화를 조정하기 위해 각 비디오의 데이터는 시간이 지남에 따라 형광 강도의 배 증가로 표현, 각각 다른 조건을 꾸몄다. 당 각 평가시기의 형광 배 증가셀은 F T는 특정 평가시기의 평균 형광이다 F T / F 0으로 계산되며, F 0 t = 0 (그림 1-2)에서의 평균 형광입니다. </li></ol>

<ol>
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저자는 더 경쟁 재정적 이해가없는 선언합니다.

기계 손상 및 세포막 수리에 대한 이전 연구는 유리 구슬, micropipetting, 전단, 긁거나 긁는을 삭제에 이르기까지, 손상 세포의 다양한 메커니즘에 의존했다. 이러한 분석법의 판독 오히려 정량보다 질적이고, 수리기구의 동역학에 정확한 정보를 제공하지 않았다. 부상 이러한 형태 후 자신의 원형질막을 봉인하는 세포의 능력은 세포막 투과성 염료, 세포질 효소 ATP 수준 또는 장기의 손실의 세포질 ...

그것은 기계적인 손상 후 세포막 수리 칼슘 2 +에 의존하는 프로세스 1,2 것을 수십 년 동안 알려져있다. 나중에 연구는 상처를 통해 칼슘 2 +의 유입이 3,4 재 밀봉에 필요한 부상의 사이트에서 세포 내 소포의 세포 외 유출의 활발한 프로세스를 트리거 것으로 나타났다. 세포의 세포질에로드 형광 염료의 손실의 비율은 수리의 속도를 평가하는 데 사용하고, 재 밀봉이 손상 후 5 &lt;30 초 이내에 완료되었다는 결론을 내렸다. 두 모델은 처음에 플라즈마 막 수리의 세포 외 유출에 대한 요구 사항을 설명하기 위해 제안했다 : 병변을 통해 칼슘 2 + 유입이 큰 &quot;패치&quot;를 형성, 세포 내 소포의 초기 호모 융합을 트리거 것을 제안 1) &quot;패치&quot;모델을하는 것 그 다음 상처 6 및 그 막 Adde 5의 제안 2) 장력 감소 모델을 봉인하는 세포막에 적용될상처의 근처에서 칼슘 2 + 의존성 세포 외 유출 d 개의 이중층 (7)의 재 밀봉을 용이하게 세포막 의욕을 감소시킬 것이다. 세포막 수리 칼슘 2 + 트리거 세포 외 유출의 역할은 상기 리소좀가 손상균 그들의 세포 외 유출 및 세포막 보수를 용이 칼슘 2 + 센서 분자 synaptotagmin VII 포함될 것을 보여주는 연구에 의해 보강되었다 8,9,10,11 . 그러나, 추가 증거만으로는 세포 외 유출이 세포막의 복구를 촉진하기에 충분하지이라고 지적했다. 원형질막에 기계적 눈물 이외에, 세포 상해의 빈번한 형태는 박테리아 12,13 또는 14,15 면역 세포에 의해 생성 된 기공 형성 독소 permeabilization이다. 기계적 눈물 달리 기공 형성 단백질은 세포막은 막을 &quot;패치&quot;를 적용함으로써 또는 현상 감소하여 단순히 재 밀봉 할 수없는 안정한 단백질 늘어선 기공 형성에 자신을 삽입막 긴장을 보내고. 흥미롭게 연구는 포유 동물 세포는 기공 형성 단백질과 permeabilization 후 자신의 세포막을 복구하는 효율적인 메커니즘을 가지고 있고,이 프로세스는 또한 세포 외 칼슘 2 + (12)의 존재를 필요로 것으로 나타났다. 기계적인 상처 (5)와 관찰이 발견은 세포 표면에서 횡단 기공 제거도 빠른 처리 여부의 문제를 제기했다. 과정은 칼슘 2 + 세포 필요로하고, 30 초 ~ 이내에 완료된다 놀랍게도, 우리의 최근의 연구는 기공 형성 독소 permeabilized 세포의 재 밀봉 기계적인 상처의 수리와 매우 유사한 특성을 가지고 것으로 나타났습니다. 이 과정을 자세히 조사하고, 우리는 최근 리소좀의 칼슘 2 + 규제 세포 외 유출뿐만 아니라, 세포막 수리 산 sphingomyelinase (ASM)을 효소 리소좀의 릴리스에 의해 트리거 필수입니다 엔도 사이토 시스의 빠른 형태를 포함 것을 알게 뿐만 아니라 일에 대한횡단 모공 전자의 제거뿐만 아니라, 기계적인 상처 (16)의 수리. 기공 형성 단백질과 permeabilization 후 세포 재 밀봉의 반응 속도를 결정하기 위해, 우리의 실험실에서 우리는 레이저 부상 고립 된 근육 섬유 (17)의 재 밀봉을 평가하기 위해 이전에 사용했던 라이브 영상 방법론을 적용. 이 분석은 안정적으로 형광 강도가 증가하고 지질 이중층의 외부 전단지에 인 인터 친 유성 염료 FM1-43의 특성에 의존한다. 세포막 이중층은 세포막의 손상을 감지하고 18,16,19,20를 복구하는 중요한 분석을 제공, 세포 내 세포막 세포 염료 이익 액세스 중단됩니다. 기공 형성 단백질과 permeabilization 후 세포 재 밀봉의 평가에이 분석을 적용하기 위해, 우리는 막 콜레스테롤 (21)에 결합하는 세균성 독소 streptolysin의 O (SLO), 4 ° C에서 세포를 미리 배양 하였다. 동기 셀permeabilization 후 쉽게 기공 형성을 횡단하는 형태의 리드 올리고머 및 변경을 활성화하는 가열 현미경 스테이지에서 매체 가온 얼음에서 세포를 이동시킴으로써 달성 될 수있다. 이 방법의 장점은, 레이저 상흔을 사용 이전에 발행 된 분석법을 통해, 셀들의 많은 수는 세포 집단의 더 나은 샘플링을 제공 현미경 분야에서 동시에 분석 될 수 있다는 점이다. 기공 형성 독소 기계적 손상과 permeabilization 후 본 셀 재 밀봉 공정의 기계적인 유사성을 감안할 때, 우리가 여기서 설명하는 분석은 세포막 수리의 기본 과정에 참여 요인을 해부에 매우 다양하고 강력한 방법을 제공한다. 예로서, 우리는 복구 프로세스의 칼슘 2 + 종속 독립 단계를 식별하는이 분석을 사용하는 것이 가능하다는 것을 보여준다.

<table border="1" fo:keep-together.within-page="always">
		<tbody>
		 <tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Reagent</td>
		 </tr>
		 <tr>
			<td>HeLa 229 cell line</td>
			<td>ATCC</td>
			<td>CCL2-1</td>
			<td></td>
		 </tr>
		 <tr>
			<td>DMEM High Glucose </td>
			<td>Invitrogen</td>
			<td>11965</td>
			<td></td>
		 </tr>
		 <tr>
			<td>DMEM High Glucose No Calcium</td>
			<td>Invitrogen</td>
			<td>20168</td>
			<td></td>
		 </tr>
		 <tr>
			<td>FM1-43</td>
			<td>Invitrogen</td>
			<td>T3163</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Optimem Reduced Serum</td>
			<td>Invitrogen</td>
			<td>31985</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Lipofectamine RNAiMax</td>
			<td>Invitrogen</td>
			<td>13778</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Control medium GC content RNAi oligo</td>
			<td>Invitrogen</td>
			<td>12935300</td>
			<td></td>
		 </tr>
		 <tr>
			<td>SMPD1 RNAi oligo</td>
			<td>Invitrogen</td>
			<td>HSS143988</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Fetal Bovine Serum Heat-inactivated</td>
			<td>Gemini-Bioproducts</td>
			<td>100-106</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Sphingomyelinase from Bacillus cereus</td>
			<td>Sigma</td>
			<td>S7651</td>
			<td></td>
		 </tr>
		 <tr>
			<td>35 mm-glass bottom dishes</td>
			<td>MatTek</td>
			<td>P35G-0-14</td>
			<td></td>
		 </tr>
		 <tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Material</td>
		 </tr>
		 <tr>
			<td>Inverted microscope</td>
			<td>Nikon</td>
			<td>Eclipse Ti</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Camera</td>
			<td>Hamamatsu Photonics</td>
			<td>C9100-50</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Spinning disk confocal microscope</td>
			<td>PerkinElmer</td>
			<td>UltraViewVoX</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Software analysis software</td>
			<td>PerkinElmer</td>
			<td>Volocity Suite</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Environmental chamber</td>
			<td>Pathology Devices</td>
			<td>LiveCell System Chamber</td>
		 </tr>
		</tbody>
 </table>

live imaging assay for assessing the roles of ca2+ and sphingomyelinase in the repair of pore-forming toxin wounds

세포막 손상은 빈번한 이벤트이며, 상처는 빠르게 세포의 생존을 보장하기 위해 복구해야 할. 칼슘 2 +의 유입은 30 초 ~ 내의 세포막에 기계적인 상처의 수리를 트리거 키 신호 이벤트입니다. 최근의 연구는 포유 동물 세포는 또한 기공은 기공 엔도 시토 시스 뒤에 리소좀 효소 산 sphingomyelinase의 세포 외 유출을 포함하는 칼슘 2 +에 의존하는 과정에서 독소를 형성하는 permeabilization 후 자신의 세포막을 봉인 것으로 나타났다. 여기, 우리는 독소 streptolysin의 O에 의해 permeabilized 세포의 재 밀봉이 칼슘 2 + 유입에도 신속하고 종속이 있음을 입증하는 데 사용되는 방법을 설명합니다. 분석 설계는 허용 부상 이벤트의 동기화 및 촬상하여 세포막 무결성을 복원하고 liphophilic 염료 FM1-43 세포의 세포막에 도달하는 정도를 정량화하는 세포의 능력의 정확한 운동 측정. 이 라이브 분석 ALSO는 외생의 능력의 중요한 평가들이 세포막을 복구하는 세포의 능력을 반영 FM1-43의 유입을 억제하는 등 sphingomyelinase 같은 가용성 인자를 첨가한다. 이 분석은 우리가 효소의 세포 또한이 칼슘 2 +의 부재에서 permeabilized 세포의 재 밀봉을 촉진하기 때문에 sphingomyelinase, 캘리포니아 2 +에 의존하는 세포 외 유출의 하류 역할을하는 것이 처음으로 보여줄 수있었습니다.

리소좀 산 sphingomyelinase 고갈 세포에서 세균 sphingomyelinase (SM) 구조 세포막 보수 저농도. FM1-43 영상 분석을 사용하여, 우리는 이전에 칼슘 2 +의 존재에 부상 리소좀 효소 ASM에 대한 결핍과 SLO에 노출 된 세포는 세포막의 결함이 재조합 인간 효소 (19)의 외 추가로 구출되는 것을 보여 주었다. 인간의 리소좀 ASM은 낮은 pH의 최적를 가지고 있기 때문에, 우리는 재 밀?...

Watch this Scientific Journal Video about Live Imaging Assay for Assessing the Roles of Ca2+ and Sphingomyelinase in the Repair of Pore-forming Toxin Wounds at JoVE.com

Live Imaging Assay for Assessing the Roles of Ca2+ and Sphingomyelinase in the Repair of Pore-forming Toxin Wounds

친 유성 염료 FM1-43에 노출 된 세포의 라이브 영상은 기공 형성 독소는 세포막에서 제거되는 반응 속도의 정밀한 결정을 허용한다. 이것은 CA에 대한 요구 사항이 +, sphingomyelinase 및 세포막 수리 및 다른 인자를 평가하는 데 사용될 수있는 민감한 분석이다.

CA의 역할을 평가하기위한 영상 분석을 라이브<sup&gt; 2 +</sup기공 형성 독소 상처의 수리에&gt;와 Sphingomyelinase

Plasma membrane injury is a frequent event, and wounds have to be rapidly repaired to ensure cellular survival. Influx of Ca2+ is a key signaling event ...

live-imaging-assay-for-assessing-roles-ca2-sphingomyelinase-repair

Research

JoVE Journal

Biology

Live Imaging Assay for Assessing the Roles of Ca2+ and Sphingomyelinase in the Repair of Pore-forming Toxin Wounds

11.6K 조회수.  University of Maryland.  친 유성 염료 FM1-43에 노출 된 세포의 라이브 영상은 기공 형성 독소는 세포막에서 제거되는 반응 속도의 정밀한 결정을 허용한다. 이것은 CA에 대한 요구 사항이 +, sphingomyelinase 및 세포막 수리 및 다른 인자를 평가하는 데 사용될 수있는 민감한 분석이다. 

비디오: Live Imaging Assay for Assessing the Roles of Ca2+ and Sphingomyelinase in the Repair of Pore-forming Toxin Wounds

The Preparation of Drosophila Embryos for Live-Imaging Using the Hanging Drop Protocol

Green fluorescent protein (GFP)-based timelapse live-imaging is a powerful technique for studying the genetic regulation of dynamic processes such as tissue morphogenesis, cell-cell adhesion, or cell death. Drosophila embryos expressing GFP are readily imaged using either stereoscopic or confocal microscopy. A goal of any live-imaging protocol is to minimize detrimental effects such as dehydration and hypoxia. Previous protocols for preparing Drosophila embryos for live-imaging analysis have involved placing dechorionated embryos in halocarbon oil and sandwiching them between a halocarbon gas-permeable membrane and a coverslip1-3. The introduction of compression through mounting embryos in this manner represents an undesirable complication for any biomechanical-based analysis of morphogenesis. Our method, which we call the hanging drop protocol, results in excellent viability of embryos during live imaging and does not require that embryos be compressed. Briefly, the hanging drop protocol involves the placement of embryos in a drop of halocarbon oil that is suspended from a coverslip, which is, in turn, fixed in position over a humid chamber. In addition to providing gas exchange and preventing dehydration, this arrangement takes advantage of the buoyancy of embryos in halocarbon oil to prevent them from drifting out of position during timelapse acquisition. This video describes in detail how to collect and prepare Drosophila embryos for live imaging using the hanging drop protocol. This protocol is suitable for imaging dechorionated embryos using stereomicroscopy or any upright compound fluorescence microscope.

The Preparation of Drosophila Embryos for Live-Imaging Using the Hanging Drop Protocol

A simple, inexpensive, and effective method of preparing Drosophila embryos for live-imaging analysis is presented. Our protocol provides humidity and gas exchange and does not compress the Drosophila embryo. This method is suitable for GFP-based live imaging of Drosophila embryos using a stereomicroscope or upright compound microscope.

의 준비 Drosophila</em공중 드롭 프로토콜을 사용하여 라이브 영상을위한> 태아

Green fluorescent protein (GFP)-based timelapse live-imaging is a powerful technique for studying the genetic regulation of dynamic processes such as ...

연구

생물학

Live Imaging of the Zebrafish Embryonic Brain by Confocal Microscopy

In this video, we demonstrate the method our lab has developed to analyze the cell shape changes and rearrangements required to bend and fold the developing zebrafish brain (Gutzman et al, 2008). Such analysis affords a new understanding of the underlying cell biology required for development of the 3D structure of the vertebrate brain, and significantly increases our ability to study neural tube morphogenesis. The embryonic zebrafish brain is shaped beginning at 18 hours post fertilization (hpf) as the ventricles within the neuroepithelium inflate. By 24 hpf, the initial steps of neural tube morphogenesis are complete. Using the method described here, embryos at the one cell stage are injected with mRNA encoding membrane-targeted green fluorescent protein (memGFP). After injection and incubation, the embryo, now between 18 and 24 hpf, is mounted, inverted, in agarose and imaged by confocal microscopy. Notably, the zebrafish embryo is transparent making it an ideal system for fluorescent imaging. While our analyses have focused on the midbrain-hindbrain boundary and the hindbrain, this method could be extended for analysis of any region in the zebrafish to a depth of 
80-100 &mu;m.

In this video, we demonstrate a method by which to analyze the developing vertebrate brain in live zebrafish embryos at single cell resolution by confocal microscopy. This includes the method by which we inject the single-cell zebrafish embryo and subsequently mount and image the developing brain.

공촛점 현미경으로 Zebrafish 배아 뇌 영상 라이브

In this video, we demonstrate the method our lab has developed to analyze the cell shape changes and rearrangements required to bend and fold the ...

Techniques for Imaging Ca2+ Signaling in Human Sperm

Fluorescence microscopy of cells loaded with fluorescent, Ca2+-sensitive dyes is used for measurement of spatial and temporal aspects of Ca2+ signaling in live cells. Here we describe the method used in our laboratories for loading suspensions of human sperm with Ca2+-reporting dyes and measuring the fluorescence signal during physiological stimulation. Motile cells are isolated by direct swim-up and incubated under capacitating conditions for 0-24 h, depending upon the experiment. The cell-permeant AM (acetoxy methyl ester) ester form of the Ca2+-reporting dye is then added to a cell aliquot and a period of 1 h is allowed for loading of the dye into the cytoplasm. We use visible wavelength dyes to minimize photo-damage to the cells, but this means that ratiometric recording is not possible. Advantages and disadvantages of this approach are discussed. During the loading period cells are introduced into an imaging chamber and allowed to adhere to a poly-D-lysine coated coverslip. At the end of the loading period excess dye and loose cells are removed by connection of the chamber to the perfusion apparatus. The chamber is perfused continuously, stimuli and modified salines are then added to the perfusion header. Experiments are recorded by time-lapse acquisition of fluorescence images and analyzed in detail offline, by manually drawing regions of interest. Data are normalized to pre-stimulus levels such that, for each cell (or part of a cell), a graph showing the Ca2+ response as % change in fluorescence is obtained.

Techniques for Imaging Ca2+ Signaling in Human Sperm

Stimulus-evoked [Ca2+]i signals of individual human sperm are assessed. Motile cells are loaded with Ca2+-sensitive fluorescent dye (AM-ester method) and immobilised in a perfusable chamber. Cells are imaged by time-lapse fluorescence microscopy and stimulated via the perfusing medium. Responses of single cells (or regions) are analysed offline using Excel.

이미징 CA의 기술 2 + 인간 정자의 시그널링

Fluorescence microscopy of cells loaded with fluorescent, Ca2+-sensitive dyes is used for measurement of spatial and temporal aspects of Ca2+ signaling in ...

Electron Spin Resonance Micro-imaging of Live Species for Oxygen Mapping

This protocol describes an electron spin resonance (ESR) micro-imaging method for three-dimensional mapping of oxygen levels in the immediate environment of live cells with micron-scale resolution1. Oxygen is one of the most important molecules in the cycle of life. It serves as the terminal electron acceptor of oxidative phosphorylation in the mitochondria and is used in the production of reactive oxygen species. Measurements of oxygen are important for the study of mitochondrial and metabolic functions, signaling pathways, effects of various stimuli, membrane permeability, and disease differentiation. Oxygen consumption is therefore an informative marker of cellular metabolism, which is broadly applicable to various biological systems from mitochondria to cells to whole organisms. Due to its importance, many methods have been developed for the measurements of oxygen in live systems. Current attempts to provide high-resolution oxygen imaging are based mainly on optical fluorescence and phosphorescence methods that fail to provide satisfactory results as they employ probes with high photo-toxicity and low oxygen sensitivity. ESR, which measures the signal from exogenous paramagnetic probes in the sample, is known to provide very accurate measurements of oxygen concentration. In a typical case, ESR measurements map the probe's lineshape broadening and/or relaxation-time shortening that are linked directly to the local oxygen concentration. (Oxygen is paramagnetic; therefore, when colliding with the exogenous paramagnetic probe, it shortness its relaxation times.) Traditionally, these types of experiments are carried out with low resolution, millimeter-scale ESR for small animals imaging. Here we show how ESR imaging can also be carried out in the micron-scale for the examination of small live samples. ESR micro-imaging is a relatively new methodology that enables the acquisition of spatially-resolved ESR signals with a resolution approaching 1 micron at room temperature2. The main aim of this protocol-paper is to show how this new method, along with newly developed oxygen-sensitive probes, can be applied to the mapping of oxygen levels in small live samples. A spatial resolution of ~30 x 30 x 100 &mu;m is demonstrated, with near-micromolar oxygen concentration sensitivity and sub-femtomole absolute oxygen sensitivity per voxel. The use of ESR micro-imaging for oxygen mapping near cells complements the currently available techniques based on micro-electrodes or fluorescence/phosphorescence. Furthermore, with the proper paramagnetic probe, it will also be readily applicable for intracellular oxygen micro-imaging, a capability which other methods find very difficult to achieve.

This protocol describes a method for micron-scale three-dimensional imaging of oxygen concentration in the immediate environment of live cells by electron spin resonance microscopy.

전자 스핀 공명 산소 매핑에 대한 라이브 종의 마이크로 이미징

This protocol describes an electron spin resonance (ESR) micro-imaging method for three-dimensional mapping of oxygen levels in the immediate environment ...

Live Cell Calcium Imaging Combined with siRNA Mediated Gene Silencing Identifies Ca2+ Leak Channels in the ER Membrane and their Regulatory Mechanisms

In mammalian cells, the endoplasmic reticulum (ER) plays a key role in protein biogenesis as well as in calcium signalling1. The heterotrimeric Sec61 complex in the ER membrane provides an aqueous path for newly-synthesized polypeptides into the lumen of the ER. Recent work from various laboratories suggested that this heterotrimeric complex may also form transient Ca2+ leak channels2-8. The key observation for this notion was that release of nascent polypeptides from the ribosome and Sec61 complex by puromycin leads to transient release of Ca2+ from the ER. Furthermore, it had been observed in vitro that the ER luminal protein BiP is involved in preventing ion permeability at the level of the Sec61 complex9,10. We have established an experimental system that allows us to directly address the role of the Sec61 complex as potential Ca2+ leak channel and to characterize its putative regulatory mechanisms11-13. This system combines siRNA mediated gene silencing and live cell Ca2+ imaging13. Cells are treated with siRNAs that are directed against the coding and untranslated region (UTR), respectively, of the SEC61A1 gene or a negative control siRNA. In complementation analysis, the cells are co-transfected with an IRES-GFP vector that allows the siRNA-resistant expression of the wildtype SEC61A1 gene. Then the cells are loaded with the ratiometric Ca2+-indicator FURA-2 to monitor simultaneously changes in the cytosolic Ca2+ concentration in a number of cells via a fluorescence microscope. The continuous measurement of cytosolic Ca2+ also allows the evaluation of the impact of various agents, such as puromycin, small molecule inhibitors, and thapsigargin on Ca2+ leakage. This experimental system gives us the unique opportunities to i) evaluate the contribution of different ER membrane proteins to passive Ca2+ efflux from the ER in various cell types, ii) characterize the proteins and mechanisms that limit this passive Ca2+ efflux, and iii) study the effects of disease linked mutations in the relevant components.

Live Cell Calcium Imaging Combined with siRNA Mediated Gene Silencing Identifies Ca2+ Leak Channels in the ER Membrane and their Regulatory Mechanisms

The endoplasmic reticulum plays a key role in protein biogenesis and in calcium homeostasis. We have established an experimental system that allows us to address the role of Ca2+ leak channels and to characterize their putative regulatory mechanisms. This system involves siRNA mediated gene silencing and live cell Ca2+ imaging.

siRNA의 중재 진 입을와 결합 라이브 세포 칼슘 영상은 CA를 나타냅니다 2 + ER 막의 누출 채널과 규제 메커니즘

In mammalian cells, the endoplasmic reticulum (ER) plays a key role in protein biogenesis as well as in calcium signalling1. The heterotrimeric Sec61 ...

An Explant Assay for Assessing Cellular Behavior of the Cranial Mesenchyme

The central nervous system is derived from the neural plate that undergoes a series of complex morphogenetic movements resulting in formation of the neural tube in a process known as neurulation. During neurulation, morphogenesis of the mesenchyme that underlies the neural plate is believed to drive neural fold elevation. The cranial mesenchyme is comprised of the paraxial mesoderm and neural crest cells. The cells of the cranial mesenchyme form a pourous meshwork composed of stellate shaped cells and intermingling extracellular matrix (ECM) strands that support the neural folds. During neurulation, the cranial mesenchyme undergoes stereotypical rearrangements resulting in its expansion and these movements are believed to provide a driving force for neural fold elevation. However, the pathways and cellular behaviors that drive cranial mesenchyme morphogenesis remain poorly studied. Interactions between the ECM and the cells of the cranial mesenchyme underly these cell behaviors. Here we describe a simple ex vivo explant assay devised to characterize the behaviors of these cells. This assay is amendable to pharmacological manipulations to dissect the signaling pathways involved and live imaging analyses to further characterize the behavior of these cells. We present a representative experiment demonstrating the utility of this assay in characterizing the migratory properties of the cranial mesenchyme on a variety of ECM components.

The cranial mesenchyme undergoes dramatic morphogenic movements that likely provides a driving force for elevation of the neural folds1,2. Here we describe a simple ex vivo explant assay to characterize the cellular behaviors of the cranial mesenchyme during neurulation. This assay has numerous applications including being amenable to pharmacological manipulations and live imaging analyses.

두개골 Mesenchyme의 셀룰러 행동을 평가하기위한 Explant 분석

The central nervous system is derived from the neural plate that undergoes a series of complex morphogenetic movements resulting in formation of the ...

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

The proper elimination of unwanted or aberrant cells through apoptosis and subsequent phagocytosis (apoptotic cell clearance) is crucial for normal development in all metazoan organisms. Apoptotic cell clearance is a highly dynamic process intimately associated with cell death; unengulfed apoptotic cells are barely seen in vivo under normal conditions. In order to understand the different steps of apoptotic cell clearance and to compare 'professional' phagocytes - macrophages and dendritic cells to 'non-professional' - tissue-resident neighboring cells, in vivo live imaging of the process is extremely valuable. Here we describe a protocol for studying apoptotic cell clearance in live Drosophila embryos. To follow the dynamics of different steps in phagocytosis we use specific markers for apoptotic cells and phagocytes. In addition, we can monitor two phagocyte systems in parallel: 'professional' macrophages and 'semi-professional' glia in the developing central nervous system (CNS). The method described here employs the Drosophila embryo as an excellent model for real time studies of apoptotic cell clearance.

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

Here we describe an effective method for studying dynamics of apoptotic cell clearance in vivo. This method employs live Drosophila embryos as a powerful model for monitoring phagocytosis of apoptotic cells using specific labeling of apoptotic cells and phagocytes.

사멸 세포 간극의 이미지를하는 동안 라이브<em&gt; 초파리</em&gt; 배아

The  proper elimination of unwanted or aberrant cells through apoptosis and  subsequent phagocytosis (apoptotic cell clearance) is crucial for normal  ...

Live Cell Imaging of Early Autophagy Events: Omegasomes and Beyond

Autophagy is a cellular response triggered by the lack of nutrients, especially the absence of amino acids. Autophagy is defined by the formation of double membrane structures, called autophagosomes, that sequester cytoplasm, long-lived proteins and protein aggregates, defective organelles, and even viruses or bacteria. Autophagosomes eventually fuse with lysosomes leading to bulk degradation of their content, with the produced nutrients being recycled back to the cytoplasm. Therefore, autophagy is crucial for cell homeostasis, and dysregulation of autophagy can lead to disease, most notably neurodegeneration, ageing and cancer.	
	Autophagosome formation is a very elaborate process, for which cells have allocated a specific group of proteins, called the core autophagy machinery. The core autophagy machinery is functionally complemented by additional proteins involved in diverse cellular processes, e.g. in membrane trafficking, in mitochondrial and lysosomal biology. Coordination of these proteins for the formation and degradation of autophagosomes constitutes the highly dynamic and sophisticated response of autophagy. Live cell imaging allows one to follow the molecular contribution of each autophagy-related protein down to the level of a single autophagosome formation event and in real time, therefore this technique offers a high temporal and spatial resolution.	
	Here we use a cell line stably expressing GFP-DFCP1, to establish a spatial and temporal context for our analysis. DFCP1 marks omegasomes, which are precursor structures leading to autophagosomes formation. A protein of interest (POI) can be marked with either a red or cyan fluorescent tag. Different organelles, like the ER, mitochondria and lysosomes, are all involved in different steps of autophagosome formation, and can be marked using a specific tracker dye. Time-lapse microscopy of autophagy in this experimental set up, allows information to be extracted about the fourth dimension, i.e. time. Hence we can follow the contribution of the POI to autophagy in space and time.

Time-lapse microscopy of fluorescently labeled autophagy markers allows monitoring of the dynamic autophagy response with high temporal resolution. Using specific autophagy and organelle markers in a combination of 3 different colors, we can follow the contribution of a protein to autophagosome formation in a robust spatial and temporal context.

초기 자식 작용 이벤트의 셀 이미징 라이브 : Omegasomes을 넘어

Autophagy is a  cellular response triggered by the lack of nutrients, especially the absence of  amino acids. Autophagy is defined by the formation of ...

The Tomato/GFP-FLP/FRT Method for Live Imaging of Mosaic Adult Drosophila Photoreceptor Cells

The Drosophila eye is widely used as a model for studies of development and neuronal degeneration. With the powerful mitotic recombination technique, elegant genetic screens based on clonal analysis have led to the identification of signaling pathways involved in eye development and photoreceptor (PR) differentiation at larval stages. We describe here the Tomato/GFP-FLP/FRT method, which can be used for rapid clonal analysis in the eye of living adult Drosophila. Fluorescent photoreceptor cells are imaged with the cornea neutralization technique, on retinas with mosaic clones generated by flipase-mediated recombination. This method has several major advantages over classical histological sectioning of the retina: it can be used for high-throughput screening and has proved an effective method for identifying the factors regulating PR survival and function. It can be used for kinetic analyses of PR degeneration in the same living animal over several weeks, to demonstrate the requirement for specific genes for PR survival or function in the adult fly. This method is also useful for addressing cell autonomy issues in developmental mutants, such as those in which the establishment of planar cell polarity is affected.

The Tomato/GFP-FLP/FRT Method for Live Imaging of Mosaic Adult Drosophila Photoreceptor Cells

The Tomato/GFP-FLP/FRT method involves visualizing mosaic photoreceptor cells in living Drosophila. It can be used to follow individual photoreceptor cell fates in the retina for days or weeks. This method is ideal for studies of retinal degeneration and neurodegenerative diseases or photoreceptor cell development.

모자이크 성인의 라이브 이미징 토마토 / GFP-FLP / FRT 방법<em&gt; 초파리</em&gt; 광 수용체 (photoreceptor)

The Drosophila eye is widely used as a model  for studies of development and neuronal degeneration. With the powerful mitotic  recombination technique, ...

Adipo-Clear: A Tissue Clearing Method for Three-Dimensional Imaging of Adipose Tissue

Adipose tissue plays a central role in energy homeostasis and thermoregulation. It is composed of different types of adipocytes, as well as adipocyte precursors, immune cells, fibroblasts, blood vessels, and nerve projections. Although the molecular control of cell type specification and how these cells interact have been increasingly delineated, a more comprehensive understanding of these adipose-resident cells can be achieved by visualizing their distribution and architecture throughout the whole tissue. Existing immunohistochemistry and immunofluorescence approaches to analyze adipose histology rely on thin paraffin-embedded sections. However, thin sections capture only a small portion of tissue; as a result, the conclusions can be biased by what portion of tissue is analyzed. We have therefore developed an adipose tissue clearing technique, Adipo-Clear, to permit comprehensive three-dimensional visualization of molecular and cellular patterns in whole adipose tissues. Adipo-Clear was adapted from iDISCO/iDISCO+, with specific modifications made to completely remove the lipid stored in the tissue while preserving native tissue morphology. In combination with light-sheet fluorescence microscopy, we demonstrate here the use of the Adipo-Clear method to obtain high-resolution volumetric images&#160;of an entire adipose tissue.

Due to the high lipid content, adipose tissue has been challenging to visualize using traditional histological methods. Adipo-Clear is a tissue clearing technique that allows robust labeling and high-resolution volumetric fluorescent imaging of adipose tissue. Here, we describe the methods for sample preparation, pretreatment, staining, clearing, and mounting for imaging.

Adipo-지우기: 조직 지방 조직의 3 차원 이미징 방법 삭제

Adipose tissue plays a central role in energy homeostasis and thermoregulation. It is composed of different types of adipocytes, as well as adipocyte ...