우리는 신경 과학 연구를위한 Tufts 센터, P30 NS047243 (잭슨), 보스턴 대학 의과 대학, 링크 의학 공단에서 학제간 바이오 메디컬 연구 에반스 센터에서 지원 Mitochondria 동질 연구 협력 (mtARC), 그리고이 작품을 지원하기위한 자이스 혈구 감사드립니다.

1. 이미지 플레이트 준비 <ol><li> 10% 표준 태아 소 혈청, 1 % 페니실린 - 스트렙토 마이신, 2 개의 MM L-글루타민, 50 μm의 2 - 메르 캅 토 에탄올, 5 밀리미터 NaHCO 3, 2 MM HEPES, 2 개의 MM pyruvic 산성, 11 MM 포도당을 포함하는 RPMI 미디어 문화 INS1 세포 80 % 합류합니다. </li><li> 0.05 %의 트립신 및 플레이트 그들 폴리-D-라이신 코팅 coverslip 바닥 이미징 플레이트 (30~40%의 합류점)를 향해 함께 INS1 셀 문화를 Trypsinize. </li><li> 번호판 60~80%의 합류점 (~ 2 일)에 도달하고 mitochondrial 매트릭스는 타겟 추가할 수 있도록 허용 (COXVIII)에 대한 adenoviral PA-GFP 24 시간 (방어 = 5). 교환 매체와 세포 이미징하기 전에 다른 2 일 성장할 수 있습니다. </li><li> 이미징의 날, 이미징 플레이트로 7-15 나노미터 TMRE을 추가하고 적어도 45 분 동안 평형. </li><li> 인큐베이터에서이 시간 순서 (무대 최고 인큐베이터 여기에 사용되고있다) 동안과 현미경 ° C에서 약 1 시간 동안 37 평형 수 있습니다. 5 % CO 2를 켭니다. </리&gt; </ol> 2. 자이스 혈구 LSM 710 공촛점 현미경을위한 이미징 매개 변수 <ol><li> 현미경 equilibrated 후 취득 탭 아래, &quot;쇼 설명서 도구&quot;를 클릭 이미징이 패널을 설치 열고 &quot;채널 모드&quot;를 선택하고 모든 &quot;프레임&quot;을 추적할 전환합니다. </li><li> 라이트 경로 패널에서 LSM 및 채널 모드를 선택합니다. 표본 아이콘에서 일하는 것은 상향, 690 + 및 MBS 488 &quot;리어&quot;, MBS를 선택합니다. 패널의 아래쪽에서 명시야 또는 DIC 채널의 시각화을 활성화하는 &quot;T-PMT&quot;를 선택합니다. </li><li> 490-540 nm의와 TMRE 색소를위한 580-700 nm의에 GFP의 염료에 대한 범위를 설정하여 빛의 경로 매개 변수를 조정 완료. </li><li> 획득 모드에서 × 512 픽셀 스캔 필드, 평균 4X를 512을 사용하고 줌 계수를 (당신이 교정 목적으로 일관성있게 유지하려는 수도 있음)를 선택합니다. </li></ol> 3. 이미징 매개 변수를 최적화 <ol><li> foc는 (1.4 NA) 100x 객관 렌즈를 계획 고차 색지움 사용phototoxicity에서 mitochondria 보호하는 할로겐 라이트가 아닌 형광등, 귀하의 세포에있는 우리. </li><li> PA-GFP는 maximally 오픈 핀홀을 사용하여 세포를 표현하고 밝은 녹색 세포를 찾기 위해 스캔을위한 화면. </li><li> PA-GFP를 활성화하고 신호가 감지기를 포화하지 않는다는 보장 이미징 매개 변수를 최적화하는 데 필요한 2 광자 레이저의 가장 낮은 전력을 찾습니다. PA-GFP 신호가 몇 Z-시리즈 TMRE 신호 colocalizes 확인하십시오. TMRE 신호의 손실은 mitochondrial 탈분극 또는 phototoxicity을 표시하고, 세포는 이러한 특성을 분석에 사용하지 않아야 전시. </li><li> PA-GFP 표현하는 셀을 찾아 줌 계수를 지정합니다. </li><li> 6 조각 (특정 Z-포커스가 각 위치에서 설정하지 않는 한이 범위 모두 10 세포를 충족해야합니다)를 수집하기 위해 Z-시리즈 범위를 설정합니다. </li><li> 각 셀 (셀 1, 셀 2 등)에 이미징 방법을 저장합니다. </li></ol> 4. 자동화 조정D 프로그램의 부분 그리고 당신이 1 시간 Mitochondrial 퓨전 분석을 위해 따르는 10 개의 셀을 지정 <ol><li> multitime 창의 왼쪽 패널에서 &quot;절약&quot;패널을 선택하고 파일이 저장됩니다 위치를 지정합니다. </li><li> &quot;취득&quot;패널에서 스캔 구성 세포 1 저장한 수집 방법을로드하고 Z 스택 상자를 선택합니다. 또한 &quot;Z 스택의 Z-한가운데 표시&quot;를 선택합니다. </li><li> &quot;블록&quot;패널에서 &quot;각각의 장소에서 하나의 블록&quot;를 선택합니다. 측정되는 각 구간에 대해 &quot;추가 블록&quot;을 클릭합니다. </li><li> &quot;타이밍&quot;패널에서 &quot;처음 위치로만에 차단하기 전에 대기 간격&quot;에서 &quot;간격을 기다&quot;및 유형 &quot;0&quot;를 선택합니다. 블록 2-4이 섹션에서 &quot;15 분&quot;을해야합니다. </li><li> 장소 패널에서 선택 &quot;위치 사이의 위치를​​로드하는 데 초점을 이동&quot;및 다중 &quot;을 선택 후와&quot;모두를 지우 편집 위치 목록 &quot;을 선택&quot; &quot;로드 또는 &#39;다음 LOC의&#39;클릭 &#39;은 LOC로 이동&#39;때 아래. 설정 검색&quot; 위치는 &quot;무대를 동력. </li><li> 깨물어어 &quot;블리치&quot;패널은 &quot;표백제&quot;상자를 클릭하고 &quot;설정&quot;에서 설정 파일을 지정 메뉴를 풀다운 등 주요 소프트웨어의 윈도우에 지정. 그런 다음 &quot;표백&quot;창에서 적절한 photoactivati​​on 방법을 저장합니다. 지역 패널에서이 특정 세포에 대한 투자 수익 (ROI)를 선택하고, 윈도우 &#39;투자 수익 (ROI) 목록에 추가할 현재 영역 &quot;을 선택하여 multitime이 투자 수익 (ROI)을 추가합니다. 풀다운 메뉴를 &#39;투자 수익 (ROI) &quot;에서 같은 위치를 선택하십시오. </li></ol> 타이밍이 제대로 작동하기 위해서는 각각의 세포는 15 분 간격을해야하는 두 가지 방법 저장해야합니다. 블록의 목록에서, 첫번째는 &quot;진짜&quot;photoactivati​​on 구성을 가지게 될 것이며, 나머지 두 광자 레이저를 사용하지 않는 &quot;모의&quot;구성을해야합니다. 첫 번째 블록도 (= 0 BKIntv) 지연이없는 유일한 블록 될 것입니다. 따라서 방법은 photoactivati​​on 스캔 다음에 한베이스 라인 스캔과 함께 시작됩니다. 블록의 나머지 900 초 BkIntv를 가지고 있고,각 시점에서 타이밍의 일관성을 유지하는 것처럼 시간 0 초 두 검사가 마련되어 있습니다. <ol start="7"><li> 시간이 지남에 따라되는 10 세포의 각각의 경우이 시퀀스를 수행합니다 : 표현 PAGFP 찾기 자사의 이미징 방법으로 세포를 저장 위치는 무대 위치를 패널 표시, 이미징 방법을 지정 주요 투자 수익 (ROI) 패널을 선택 구체적인 투자 수익 (ROI)을 패널 저장하고 또한 투자 수익 (ROI)을 상자에 넣어로드 photoactivated 블리치로 관심 지역 : 투자 수익 (ROI)을 지우고 1로 스캔 확대를 재설정 다음 셀을 찾아 줌으로 설정 다음 9 세포에 대한 과정을 반복 </li></ol> 각 단계의 위치와 모든 블록 관련된 적절한 이미징 방법 (스캔 구성)가 있는지 확인합니다. 또한, 나머지는 &quot;모의&quot;방법을 포함하면서 각 위치에서 첫 번째 블록은 적절한 photoactivati​​on 메서드에 해당하는지 확인하십시오. 마지막으로, &quot;실행&quot;을 선택하십시오. 5. PA-GFP 신호 강도를 분석 <ol><lI&gt; photoactivatable-GFP 신호가 빨간색 TMRE 신호로 colocalizes있는 세포의 경우 PAGFP 이미지 (여기서 우리가 metamorph을 사용)의 배경을 뺍니다. </li><li> 그런 다음 엑셀로 데이터를 내보낼하고 각 시점에서 Z-스택에 대한 평균 강도를 계산합니다. 신호가없는 광학 부분을 폐기하십시오. </li><li> 원래 photoactivated 신호의 비율을 계산하여 각 Z-스택의 신호 희석을 측정합니다. </li></ol> 각 데이터 포인트는 각 시점에 대한 중복 Z-스택 정보를 그 결과 두 번 스캔되어 기록됩니다. Z-스택 값을 동의 확인하십시오. 그렇지 않다면,이 문제 (예 : 초점, 세포 운동)을 나타내는 것입니다. 6. 대표 결과 융합 이벤트가 활성 및 비 활성 PA-GFP mitochondrion 사이에 발생하면 mitochondrial 매트릭스 내에서 PAGFP가 아닌 라벨이 매트릭스와 혼합하고 큰 지역에 희석되고, 신호를 감소강도 (그림 1A). mitochondrial 융합의 평균이 (약 1 시간)에 도달되기 전에 INS1 세포에서 신호 강도가 크게 감소는 15 분 간격으로 발생합니다. 그림 1B의 세포 PA-GFP 및 TMRE 신호의 거의 완전한 colocalization을 전시하고 있습니다. 이러한 assays에서 TMRE의 매우 낮은 농도 (15 NM) PAGFP의 photoactivati​​on을 타겟팅하기 위해, 또한 세포의 건강을 모니터링하는 데 사용됩니다. 이 중 phototoxicity, 또는 죽어가는 상태의 세포를 원하셨기에 depolarized mitochondria의 풍부한 세포는 PAGFP 및 TMRE의 불완전한 colocalization을 주어 분석해서는 안됩니다. ~ mitochondrial 볼륨의 15 %가 활성화될 때 mitochondrial 융합을위한 평균 시간은, 보통 INS1 세포에서 1 시간입니다. 때로는 작은 면적가 켜져있는 경우에도 mitochondria 대부분 인해 더욱 융합 감지하기 어려운 경우에는 높은, 네트워크받는 것에 photoactivated되기. 다른 세포 유형은 서로 다른 평균 시간을 나타내는 수 있고 mitochondrial 역학 성격 짧은 간격과 긴 기간 동안 테스트해야합니다. mitochondrial 융합을 억제하기 위해 세포 lipotoxic 환경에서 배치될 수 있습니다. 그것은 이전에 0.4 MM은 파편이 mitochondria palmitate 것을 입증하고, mitochondrial 융합 9 억제되었습니다. 이 효과는 mitochondria가 조각되어 그림 2에서 볼 수 있지만, mtPAGFP의 신호 강도가 정상 상태 (그림 1)에서만큼 변경되지 않습니다. 따라서 PA-GFP 신호의 희석 오히려 분열보다 mitochondrial 융합으로 인해가 될 가능성이 높습니다. 다른 세포 유형에서 우리는 그러한 mitochondrial 퓨전 14 필요한 OPA1 입을 등 mitochondrial 분열을 유도하기 위해 알려진 다른 방법을 사용하는 것이 좋습니다. <img alt="그림 1" src="/files/ftp_upload/3991/3991fig1.jpg" /> 1 그림. </stroNG&gt; mitochondrial 융합 분석 중에 photoactivation 후 PA-GFP 신호의 일반적인 희석. A. PA-GFP는 점차적으로 주차이 투사된 이미지에서 볼 수 있듯이 증가 면적 이상의 단백질의 희석에 이르는 mitochondrial 퓨전 이벤트로 인해됩니다 6 광학 섹션은 15 분 간격으로 계량의. PA-GFP와 B. TMRE는 mitochondria가 depolarized 활성화되어 있지 않다는 것을 보여줍니다. <a href="http://www.jove.com/files/ftp_upload/3991/3991fig1large.jpg" target="_blank">큰 그림을 보려면 여기를 누르십시오</a> . <img alt="그림 2" src="/files/ftp_upload/3991/3991fig2.jpg" /> . Mitochondrial 융합은 0.4 밀리미터 palmitate와 저해 그림 2 PA-GFP의 희석을 감소시킵니다. 시간이 지남에 변함없는 신호 강도와 짧은 mitochondria를 보여주는 6 광학 섹션 A. 추정치. TMRE와 PA-GFP의 바실러스 Colocalization 그 mitochondria가 없습니다 보여줍니다 드편광. <a href="http://www.jove.com/files/ftp_upload/3991/3991fig2large.jpg" target="_blank">큰 그림을 보려면 여기를 누르십시오</a> .

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H., Lippincott-Schwartz, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+photoactivatable+GFP+for+selective+photolabeling+of+proteins+and+cells.">A photoactivatable GFP for selective photolabeling of proteins and cells.</a> Science. 297, 1873-1877 (2002).</li><li>Schauss, A. C., Huang, H., Choi, S. Y., Xu, L., Soubeyrand, S., Bilodeau, P., Zunino, R., Rippstein, P., Frohman, M. A., McBride, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+cell-free+mitochondrial+fusion+assay+amenable+for+high-throughput+screenings+of+fusion+modulators.">A novel cell-free mitochondrial fusion assay amenable for high-throughput screenings of fusion modulators.</a> BMC Biol. 8, 100 (2011).</li><li>Sheridan, C., Martin, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+fission/fusion+dynamics+and+apoptosis.">Mitochondrial fission/fusion dynamics and apoptosis.</a> Mitochondrion. 10, 640-648 (2010).</li><li>Twig, G., Elorza, A., Molina, A. J., Mohamed, H., Wikstrom, J. D., Walzer, G., Stiles, L., Haigh, S. E., Katz, S., Las, G., Alroy, J., Wu, M., Py, B. F., Yuan, J., Deeney, J. T., Corkey, B. E., Shirihai, O. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fission+and+selective+fusion+govern+mitochondrial+segregation+and+elimination+by+autophagy.">Fission and selective fusion govern mitochondrial segregation and elimination by autophagy.</a> EMBO J. 27, 433-446 (2008).</li><li>Twig, G., Graf, S. A., Wikstrom, J. D., Mohamed, H., Haigh, S. E., Elorza, A., Deutsch, M., Zurgil, N., Reynolds, N., Shirihai, O. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tagging+and+tracking+individual+networks+within+a+complex+mitochondrial+web+with+photoactivatable+GFP.">Tagging and tracking individual networks within a complex mitochondrial web with photoactivatable GFP.</a> Am. J. Physiol. Cell Physiol. 291, C176-C184 (2006).</li><li>Twig, G., Hyde, B., Shirihai, O. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+fusion,+fission+and+autophagy+as+a+quality+control+axis:+the+bioenergetic+view.">Mitochondrial fusion, fission and autophagy as a quality control axis: the bioenergetic view.</a> Biochim. Biophys. Acta. 1777, 1092-1097 (2008).</li><li>Twig, G., Liu, X., Liesa, M., Wikstrom, J. D., Molina, A. J., Las, G., Yaniv, G., Hajnoczky, G., Shirihai, O. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biophysical+properties+of+mitochondrial+fusion+events+in+pancreatic+beta-cells+and+cardiac+cells+unravel+potential+control+mechanisms+of+its+selectivity.">Biophysical properties of mitochondrial fusion events in pancreatic beta-cells and cardiac cells unravel potential control mechanisms of its selectivity.</a> Am. J. Physiol. Cell. Physiol. 299, C477-C487 (2010).</li><li>Twig, G., Shirihai, O. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+interplay+between+mitochondrial+dynamics+and+mitophagy.">The interplay between mitochondrial dynamics and mitophagy.</a> Antioxid. Redox Signal. 14, 1939-1951 (2011).</li></ol>

이 문서에 대한 생산 및 무료 액세스가 자이스 혈구, 주식 회사가 후원합니다

인수 photoactivati​​on 후 15 분마다 발생하는 경우이 방법은 한 번에 약 10 세포 이미징을 허용합니다. 세포의 정확한 숫자는 하나 mtPAGFP 표현 문화 접시 내에서 세포, 그리고 얼마나 빨리 하나가 모든 소프트웨어 매개 변수를 설정할 수를 찾아 표시할 수있게하는 속도에 따라 다릅니다. 지정된 Z-스택 마진은 모든 세포를 신청하기 때문에 자동화가 원활하게 실행하기 위해서 세포의도 레이어를 사용해야합니다. 이 초기 photoactivati​​on 영역의 크기는 평균 시간을 적용합니다. mitochondrial 융합을 측정할 수있게하기 위해서는 네트워크의 나머지 부분에 대한 신호의 확산은 시간이 지남에 따라 모니터링할 수 있도록 네트워크의 유일한 10~20%를 photoactivate하는 것이 중요합니다. 네트워크의 너무 많이가 photoactivated 경우, 그것은 완전한 융합도 빠르게 발생 가능성이 있으며, 이벤트가 캡처되지 않습니다. 익스 트림주의로 이동합니다두 광자 레이저의 레이저 파워뿐만 아니라 mitochondrial 탈분극로 연결 phototoxicity을 피하기 위해 TMRE 농도를 조정합니다. mtPAGFP 신호가 TMRE 신호로 colocalizes 수 있도록하는 것은 phototoxicity과 일반 세포 건강 8,15를 평가하는 데 유용합니다. epifluoerescent 빛이있는 조명은 피해야한다. mtPAGFP을 표현 세포를 검색하는 동안 저전력 488nm의 여기로 스캔하는 동안, 핀홀이 maximally 열려 있어야합니다. PA-GFP는 1 시간 이상 신호를 측정할 수 있지만 세포 중 8 까다롭습 수 oversaturate하는 데 충분하지 photoactivate하는 두 광자 레이저 파워 조절. 일단 자동으로 프로그램이 시작되기 때문에 그러나 시간이 최적화 단계에서 지출되어야합니다 그것은 세포를 선택하여, 그것을 중지하고 재개하는 지루한이다. 품질 관리의 경우 차등 간섭 대비 (DIC)의 인수 세포에 초점을 모니터 이미지 (또는 전송 빛) 매우 될 수 있습니다도움이 또한 스캔하는 동안 침지 기름에 형성된 거품을 감지하는 좋은 방법, 이것은 때로는 무대의 움직임에서 발생합니다. 이 mtPAGFP 방법을 사용하면 분류되지 않은 사람들에게 photoactivated mitochondria에서 mitochondrial 매트릭스 단백질의 단방향 운동에서 데이터를 수집하지만, 그것은 다른 프로세스를 연구하기 위해이 기술을 활용하는 생각할 수있다. 예를 들어, 특정 fluorochromes는 ABC-날 15 가용성 매트릭스 단백질의 혼합에서 다른 시간 척도에서 발생하는의 융합에 표시되었습니다대로 퓨전 이벤트 기간 동안 자신의 구체적인 움직임을 관찰하는 멤브레인 단백질에 첨부하실 수 있습니다.

<table border="1"><tbody><tr><td> 시약의 이름 </td><td> 회사 </td><td> 카탈로그 번호 </td></tr><tr><td> COXIII-adenoviral PA-GFP </td><td> 박사 리핀코트요 - 슈워츠 </td><td></td></tr><tr><td> TMRE </td><td> Invitrogen </td><td> T669 </td></tr><tr><td> 자이스 혈구 LSM 710 공촛점 </td><td> 자이스 혈구 </td><td></td></tr></tbody></table>

a faster, high resolution, mtpa-gfp-based mitochondrial fusion assay acquiring kinetic data of multiple cells in parallel using confocal microscopy

Mitochondrial fusion plays an essential role in mitochondrial calcium homeostasis, bioenergetics, autophagy and quality control. Fusion is quantified in living cells by photo-conversion of matrix targeted photoactivatable GFP (mtPAGFP) in a subset of mitochondria. The rate at which the photoconverted molecules equilibrate across the entire mitochondrial population is used as a measure of fusion activity. Thus far measurements were performed using a single cell time lapse approach, quantifying the equilibration in one cell over an hour. Here, we scale up and automate a previously published live cell method based on using mtPAGFP and a low concentration of TMRE (15 nm). This method involves photoactivating a small portion of the mitochondrial network, collecting highly resolved stacks of confocal sections every 15 min for 1 hour, and quantifying the change in signal intensity. Depending on several factors such as ease of finding PAGFP expressing cells, and the signal of the photoactivated regions, it is possible to collect around 10 cells within the 15 min intervals. This provides a significant improvement in the time efficiency of this assay while maintaining the highly resolved subcellular quantification as well as the kinetic parameters necessary to capture the detail of mitochondrial behavior in its native cytoarchitectural environment.
Mitochondrial dynamics play a role in many cellular processes including respiration, calcium regulation, and apoptosis1,2,3,13. The structure of the mitochondrial network affects the function of mitochondria, and the way they interact with the rest of the cell. Undergoing constant division and fusion, mitochondrial networks attain various shapes ranging from highly fused networks, to being more fragmented. Interestingly, Alzheimer's disease, Parkinson's disease, Charcot Marie Tooth 2A, and dominant optic atrophy have been correlated with altered mitochondrial morphology, namely fragmented networks4,10,13. Often times, upon fragmentation, mitochondria become depolarized, and upon accumulation this leads to impaired cell function18. Mitochondrial fission has been shown to signal a cell to progress toward apoptosis. It can also provide a mechanism by which to separate depolarized and inactive mitochondria to keep the bulk of the network robust14. Fusion of mitochondria, on the other hand, leads to sharing of matrix proteins, solutes, mtDNA and the electrochemical gradient, and also seems to prevent progression to apoptosis9. How fission and fusion of mitochondria affects cell homeostasis and ultimately the functioning of the organism needs further understanding, and therefore the continuous development and optimization of how to gather information on these phenomena is necessary.
Existing mitochondrial fusion assays have revealed various insights into mitochondrial physiology, each having its own advantages. The hybrid PEG fusion assay7, mixes two populations of differently labeled cells (mtRFP and mtYFP), and analyzes the amount of mixing and colocalization of fluorophores in fused, multinucleated, cells. Although this method has yielded valuable information, not all cell types can fuse, and the conditions under which fusion is stimulated involves the use of toxic drugs that likely affect the normal fusion process. More recently, a cell free technique has been devised, using isolated mitochondria to observe fusion events based on a luciferase assay1,5. Two human cell lines are targeted with either the amino or a carboxy terminal part of Renilla luciferase along with a leucine zipper to ensure dimerization upon mixing. Mitochondria are isolated from each cell line, and fused. The fusion reaction can occur without the cytosol under physiological conditions in the presence of energy, appropriate temperature and inner mitochondrial membrane potential. Interestingly, the cytosol was found to modulate the extent of fusion, demonstrating that cell signaling regulates the fusion process 4,5. This assay will be very useful for high throughput screening to identify components of the fusion machinery and also pharmacological compounds that may affect mitochondrial dynamics. However, more detailed whole cell mitochondrial assays will be needed to complement this in vitro assay to observe these events within a cellular environment.
A technique for monitoring whole-cell mitochondrial dynamics has been in use for some time and is based on a mitochondrially-targeted photoactivatable GFP (mtPAGFP)6,11. Upon expression of the mtPAGFP, a small portion of the mitochondrial network is photoactivated (10-20%), and the spread of the signal to the rest of the mitochondrial network is recorded every 15 minutes for 1 hour using time lapse confocal imaging. Each fusion event leads to a dilution of signal intensity, enabling quantification of the fusion rate. Although fusion and fission are continuously occurring in cells, this technique only monitors fusion as fission does not lead to a dilution of the PAGFP signal6. Co-labeling with low levels of TMRE (7-15 nM in INS1 cells) allows quantification of the membrane potential of mitochondria. When mitochondria are hyperpolarized they uptake more TMRE, and when they depolarize they lose the TMRE dye. Mitochondria that depolarize no longer have a sufficient membrane potential and tend not to fuse as efficiently if at all. Therefore, active fusing mitochondria can be tracked with these low levels of TMRE9,15. Accumulation of depolarized mitochondria that lack a TMRE signal may be a sign of phototoxicity or cell death. Higher concentrations of TMRE render mitochondria very sensitive to laser light, and therefore great care must be taken to avoid overlabeling with TMRE. If the effect of depolarization of mitochondria is the topic of interest, a technique using slightly higher levels of TMRE and more intense laser light can be used to depolarize mitochondria in a controlled fashion (Mitra and Lippincott-Schwartz, 2010). To ensure that toxicity due to TMRE is not an issue, we suggest exposing loaded cells (3-15 nM TMRE) to the imaging parameters that will be used in the assay (perhaps 7 stacks of 6 optical sections in a row), and assessing cell health after 2 hours. If the mitochondria appear too fragmented and cells are dying, other mitochondrial markers, such as dsRED or Mitotracker red could be used instead of TMRE.
The mtPAGFP method has revealed details about mitochondrial network behavior that could not be visualized using other methods. For example, we now know that mitochondrial fusion can be full or transient, where matrix content can mix without changing the overall network morphology. Additionally, we know that the probability of fusion is independent of contact duration and organelle dimension, is influenced by organelle motility, membrane potential and history of previous fusion activity8,15,16,17.
In this manuscript, we describe a methodology for scaling up the previously published protocol using mtPAGFP and 15nM TMRE8 in order to examine multiple cells at a time and improve the time efficiency of data collection without sacrificing the subcellular resolution. This has been made possible by the use of an automated microscope stage, and programmable image acquisition software. Zen software from Zeiss allows the user to mark and track several designated cells expressing mtPAGFP. Each of these cells can be photoactivated in a particular region of interest, and stacks of confocal slices can be monitored for mtPAGFP signal as well as TMRE at specified intervals. Other confocal systems could be used to perform this protocol provided there is an automated stage that is programmable, an incubator with CO2, and a means by which to photoactivate the PAGFP; either a multiphoton laser, or a 405 nm diode laser.

Watch this Scientific Journal Video about A Faster, High Resolution, mtPA-GFP-based Mitochondrial Fusion Assay Acquiring Kinetic Data of Multiple Cells in Parallel Using Confocal Microscopy at JoVE.co

A Faster, High Resolution, mtPA-GFP-based Mitochondrial Fusion Assay Acquiring Kinetic Data of Multiple Cells in Parallel Using Confocal Microscopy

Mitochondrial 융합은 시간이 지남에 mitochondrial 네트워크 photoconverted 매트릭스 타겟 GFP의 평균 추적에 의해 측정되었다. 지금까지 하나의 세포는 한 번에 한 시간 동안 운동 분석을 받다 수 있습니다. 우리는 동시에이를 통해 데이터 수집 과정을 속도, 여러 개의 세포를 측정하는 방법을 제시한다.

병렬 공촛점 현미경을 사용하여 여러 세포의 운동 데이터를 취득 빠르고 높은 해상도, mtPA-GFP 기반 Mitochondrial 퓨전 분석

Mitochondrial fusion plays an essential role in mitochondrial calcium homeostasis, bioenergetics, autophagy and quality control. Fusion is quantified in ...

a-faster-high-resolution-mtpa-gfp-based-mitochondrial-fusion-assay

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Biology

16.4K 조회수.  Tufts School of Medicine. Mitochondrial 융합은 시간이 지남에 mitochondrial 네트워크 photoconverted 매트릭스 타겟 GFP의 평균 추적에 의해 측정되었다. 지금까지 하나의 세포는 한 번에 한 시간 동안 운동 분석을 받다 수 있습니다. 우리는 동시에이를 통해 데이터 수집 과정을 속도, 여러 개의 세포를 측정하는 방법을 제시한다.

비디오: A Faster, High Resolution, mtPA-GFP-based Mitochondrial Fusion Assay Acquiring Kinetic Data of Multiple Cells in Parallel Using Confocal Microscopy

Fluorescence-based Measurement of Store-operated Calcium Entry in Live Cells: from Cultured Cancer Cell to Skeletal Muscle Fiber

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to replenish depleted endoplasmic reticulum (ER) or sarcoplasmic reticulum (SR) Ca2+ stores1,2. Since Ca2+ is a ubiquitous second messenger, it is not surprising to see that SOCE plays important roles in a variety of cellular processes, including proliferation, apoptosis, gene transcription and motility. Due to its wide occurrence in nearly all cell types, including epithelial cells and skeletal muscles, this pathway has received great interest3,4. However, the heterogeneity of SOCE characteristics in different cell types and the physiological function are still not clear5-7.
The functional channel properties of SOCE can be revealed by patch-clamp studies, whereas a large body of knowledge about this pathway has been gained by fluorescence-based intracellular Ca2+ measurements because of its convenience and feasibility for high-throughput screening. The objective of this report is to summarize a few fluorescence-based methods to measure the activation of SOCE in monolayer cells, suspended cells and muscle fibers5,8-10. The most commonly used of these fluorescence methods is to directly monitor the dynamics of intracellular Ca2+ using the ratio of F340nm and F380nm (510 nm for emission wavelength) of the ratiometric Ca2+ indicator Fura-2. To isolate the activity of unidirectional SOCE from intracellular Ca2+ release and Ca2+ extrusion, a Mn2+ quenching assay is frequently used. Mn2+ is known to be able to permeate into cells via SOCE while it is impervious to the surface membrane extrusion processes or to ER uptake by Ca2+ pumps due to its very high affinity with Fura-2. As a result, the quenching of Fura-2 fluorescence induced by the entry of extracellular Mn2+ into the cells represents a measurement of activity of SOCE9. Ratiometric measurement and the Mn+2 quenching assays can be performed on a cuvette-based spectrofluorometer in a cell population mode or in a microscope-based system to visualize single cells. The advantage of single cell measurements is that individual cells subjected to gene manipulations can be selected using GFP or RFP reporters, allowing studies in genetically modified or mutated cells. The spatiotemporal characteristics of SOCE in structurally specialized skeletal muscle can be achieved in skinned muscle fibers by simultaneously monitoring the fluorescence of two low affinity Ca2+ indicators targeted to specific compartments of the muscle fiber, such as Fluo-5N in the SR and Rhod-5N in the transverse tubules9,11,12.

The extent of store-operated Ca2+ entry (SOCE) can be monitored using fluorescent Ca2+ indicators. Mn2+ quenching of such indicators assays SOCE in cultured cells and skeletal muscle fibers. A technique allowing spatial and temporal resolution of SOCE by confocal imaging of mechanically skinned muscle fibers is also described.

라이브 세포의 저장소로 작동하는 칼슘 항목의 형광 기반의 측정 : 교양 암 세포에서 골격근 섬유에

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to ...

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생물학

Analysis of SNARE-mediated Membrane Fusion Using an Enzymatic Cell Fusion Assay

The interactions of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins on vesicles (v-SNAREs) and on target membranes (t-SNAREs) catalyze intracellular vesicle fusion1-4. Reconstitution assays are essential for dissecting the mechanism and regulation of SNARE-mediated membrane fusion5. In a cell fusion assay6,7, SNARE proteins are expressed ectopically at the cell surface. These "flipped" SNARE proteins drive cell-cell fusion, demonstrating that SNAREs are sufficient to fuse cellular membranes. Because the cell fusion assay is based on microscopic analysis, it is less efficient when used to analyze multiple v- and t-SNARE interactions quantitatively.
Here we describe a new assay8 that quantifies SNARE-mediated cell fusion events by activated expression of &beta;-galactosidase. Two components of the Tet-Off gene expression system9 are used as a readout system: the tetracycline-controlled transactivator (tTA) and a reporter plasmid that encodes the LacZ gene under control of the tetracycline-response element (TRE-LacZ). We transfect tTA into COS-7 cells that express flipped v-SNARE proteins at the cell surface (v-cells) and transfect TRE-LacZ into COS-7 cells that express flipped t-SNARE proteins at the cell surface (t-cells). SNARE-dependent fusion of the v- and t-cells results in the binding of tTA to TRE, the transcriptional activation of LacZ and expression of &beta;-galactosidase. The activity of &beta;-galactosidase is quantified using a colorimetric method by absorbance at 420 nm.
The vesicle-associated membrane proteins (VAMPs) are v-SNAREs that reside in various post-Golgi vesicular compartments10-15. By expressing VAMPs 1, 3, 4, 5, 7 and 8 at the same level, we compare their membrane fusion activities using the enzymatic cell fusion assay. Based on spectrometric measurement, this assay offers a quantitative approach for analyzing SNARE-mediated membrane fusion and for high-throughput studies.

We have developed a cell fusion assay that quantifies SNARE-mediated membrane fusion events by activated expression of &beta;-galactosidase.

효소 세포 융합 분석을 사용하여 올가미로 인한 막 퓨전 분석

The interactions of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor)  proteins on vesicles (v-SNAREs) and on target membranes ...

In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy

We developed and validated a fluorescent marking methodology for clonal tracking of hematopoietic stem and progenitor cells (HSPCs) with high spatial and temporal resolution to study in vivo hematopoiesis using the murine bone marrow transplant experimental model. Genetic combinatorial marking using lentiviral vectors encoding fluorescent proteins (FPs) enabled cell fate mapping through advanced microscopy imaging. Vectors encoding five different FPs: Cerulean, EGFP, Venus, tdTomato, and mCherry were used to concurrently transduce HSPCs, creating a diverse palette of color marked cells. Imaging using confocal/two-photon hybrid microscopy enables simultaneous high resolution assessment of uniquely marked cells and their progeny in conjunction with structural components of the tissues. Volumetric analyses over large areas reveal that spectrally coded HSPC-derived cells can be detected non-invasively in various intact tissues, including the bone marrow (BM), for extensive periods of time following transplantation. Live studies combining video-rate multiphoton and confocal time-lapse imaging in 4D demonstrate the possibility of dynamic cellular and clonal tracking in a quantitative manner.

In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy

Combinatorial 5 fluorescent proteins marking of hematopoietic stem and progenitor cells allows in vivo clonal tracking via confocal and two-photon microscopy, providing insights into bone marrow hematopoietic architecture during regeneration. This method allows non-invasive fate mapping of spectrally-coded HSPCs-derived cells in intact tissues for extensive periods of time following transplantation.

공 초점 및 다중 광자 현미경을 사용하여 다섯 형광 단백질에 의해 표시됨 조혈 줄기 및 전구 세포의 생체 내 클론 추적에서

We developed and validated a fluorescent marking methodology for clonal tracking of hematopoietic stem and progenitor cells (HSPCs) with high spatial and ...

High-resolution Time-lapse Imaging and Automated Analysis of Microtubule Dynamics in Living Human Umbilical Vein Endothelial Cells

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes within individual endothelial cells (ECs). These processes are critical for homeostatic maintenance such as wound healing, and are also crucial in promoting tumor growth and metastasis. EC morphology is defined by the organization of the cytoskeleton, a tightly regulated system of actin and microtubule (MT) dynamics that is known to control EC branching, polarity and directional migration, essential components of angiogenesis. To study MT dynamics, we used high-resolution fluorescence microscopy coupled with computational image analysis of fluorescently-labeled MT plus-ends to investigate MT growth dynamics and the regulation of EC branching morphology and directional migration. Time-lapse imaging of living Human Umbilical Vein Endothelial Cells (HUVECs) was performed following transfection with fluorescently-labeled MT End Binding protein 3 (EB3) and Mitotic Centromere Associated Kinesin (MCAK)-specific cDNA constructs to evaluate effects on MT dynamics. PlusTipTracker software was used to track EB3-labeled MT plus ends in order to measure MT growth speeds and MT growth lifetimes in time-lapse images. This methodology allows for the study of MT dynamics and the identification of how localized regulation of MT dynamics within sub-cellular regions contributes to the angiogenic processes of EC branching and migration.

Protocols for Human Umbilical Vein Endothelial Cell (HUVEC) culture, transient transfection of fluorescently-labeled markers of microtubule growth, live-cell imaging and automated analysis of interphase microtubule growth dynamics are detailed.

고해상도 시간 경과 이미징 및 생활 인간 제대 정맥 내피 세포의 미세 소관 역학의 자동 분석

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes ...

High-resolution Respirometry to Assess Mitochondrial Function in Permeabilized and Intact Cells

A high-resolution oxygraph is a device for measuring cellular oxygen consumption in a closed-chamber system with very high resolution and sensitivity in biological samples (intact and permeabilized cells, tissues or isolated mitochondria). The high-resolution oxygraph device is equipped with two chambers and uses polarographic oxygen sensors to measure oxygen concentration and calculate oxygen consumption within each chamber. Oxygen consumption rates are calculated using software and expressed as picomoles per second per number of cells. Each high-resolution oxygraph chamber contains a stopper with injection ports, which makes it ideal for substrate-uncoupler-inhibitor titrations or detergent titration protocols for determining effective and optimum concentrations for plasma membrane permeabilization. The technique can be applied to measure respiration in a wide range of cell types and also provides information on mitochondrial quality and integrity, and maximal mitochondrial respiratory electron transport system capacity.

High-resolution respirometry is used to determine mitochondrial oxygen consumption. This is a straightforward technique to determine mitochondrial respiratory chain complexes&#39; (I-IV) respiratory rates, maximal mitochondrial electron transport system capacity, and mitochondrial outer membrane integrity.

고해상도 호흡율은 투과성이 그대로 세포에서 미토콘드리아 기능을 평가하는

A high-resolution oxygraph is a device for measuring cellular oxygen consumption in a closed-chamber system with very high resolution and sensitivity in ...

Combining Mitotic Cell Synchronization and High Resolution Confocal Microscopy to Study the Role of Multifunctional Cell Cycle Proteins During Mitosis

Study of the various regulatory events of the cell cycle in a phase-dependent manner provides a clear understanding about cell growth and division. The synchronization of cell populations at specific stages of the cell cycle has been found to be very useful in such experimental endeavors. Synchronization of cells by treatment with chemicals that are relatively less toxic can be advantageous over the use of pharmacological inhibitory drugs for the study of consequent cell cycle events and to obtain specific enrichment of selected mitotic stages. Here, we describe the protocol for synchronizing human cells at different stages of the cell cycle, including both in S phase and M phase with a double thymidine block and release procedure for studying the functionality of mitotic proteins in chromosome alignment and segregation. This protocol has been extremely useful for studying the mitotic roles of multifunctional proteins which possess established interphase functions. In our case, the mitotic role of Cdt1, a protein critical for replication origin licensing in G1 phase, can be studied effectively only when G2/M-specific Cdt1 can be depleted. We describe the detailed protocol for depletion of G2/M-specific Cdt1 using double thymidine synchronization. We also explain the protocol of cell fixation, and live cell imaging using high resolution confocal microscopy after thymidine release. The method is also useful for analyzing the function of mitotic proteins under both physiological and perturbed conditions such as for Hec1, a component of the Ndc80 complex, as it enables one to obtain large sample sizes of mitotic cells for fixed and live cell analysis as we show here.&#160;&#160;

We present a protocol for double thymidine synchronization of HeLa cells followed by analysis using high resolution confocal microscopy. This method is key to obtaining large number of cells that proceed synchronously from S phase to mitosis, enabling studies on mitotic roles of multifunctional proteins which also possess interphase functions.

유사 분열 동안 다기능 세포 주기 단백질의 역할을 연구 하기 Mitotic 세포 동기화와 고해상도 Confocal 현미경 검사 법

Study of the various regulatory events of the cell cycle in a phase-dependent manner provides a clear understanding about cell growth and division. The ...

High-resolution Respirometry to Measure Mitochondrial Function of Intact Beta Cells in the Presence of Natural Compounds

High-resolution respirometry allows for the measurement of oxygen consumption of isolated mitochondria, cells and tissues. Beta cells play a critical role in the body by controlling blood glucose levels through insulin secretion in response to elevated glucose concentrations. Insulin secretion is controlled by glucose metabolism and mitochondrial respiration. Therefore, measuring intact beta cell respiration is essential to be able to improve beta cell function as a treatment for diabetes. Using intact 832/13 INS-1 derived beta cells we can measure the effect of increasing glucose concentration on cellular respiration. This protocol allows us to measure beta cell respiration in the presence or absence of various compounds, allowing one to determine the effect of given compounds on intact cell respiration. Here we demonstrate the effect of two naturally occurring compounds, monomeric epicatechin and curcumin, on beta cell respiration under the presence of low (2.5 mM) or high glucose (16.7 mM) conditions. This technique can be used to determine the effect of various compounds on intact beta cell respiration in the presence of differing glucose concentrations.

The goal of this protocol is to measure the effect of glucose-mediated changes to mitochondrial respiration in the presence of natural compounds on intact 832/13 beta cells using high-resolution respirometry.

고해상도 Respirometry 천연 화합물 존재 그대로 베타 세포의 미토 콘 드리 아 기능을 측정 하

High-resolution respirometry allows for the measurement of oxygen consumption of isolated mitochondria, cells and tissues. Beta cells play a critical role ...

Parallel High Throughput Single Molecule Kinetic Assay for Site-Specific DNA Cleavage

Site-specific DNA cleavage (SSDC) is a key step in many cellular processes, and it is crucial to gene editing. This work describes a kinetic assay capable of measuring SSDC in many single DNA molecules simultaneously. Bead-tethered substrate DNAs, each containing a single copy of the target sequence, are prepared in a microfluidic flow channel. An external magnet applies a weak force to the paramagnetic beads. The integrity of up to 1,000 individual DNAs can be monitored by visualizing the microbeads under darkfield imaging using a wide-field, low magnification objective. Injecting of a restriction endonuclease, NdeI, initiates the cleavage reaction. Video microscopy is used to record the exact moment of each DNA cleavage by observing the frame in which the associated bead moves up and out of the focal plane of the objective. Frame-by-frame bead counting quantifies the reaction, and an exponential fit determines the reaction rate. This method allows collection of quantitative and statistically significant data on single molecule SSDC reactions in a single experiment.

A highly parallel method for measuring the site-specific cleavage of DNA at the single molecule level is described. This protocol demonstrates the technique using the restriction endonuclease NdeI. The method can easily be modified to study any process that results in site-specific DNA cleavage.

현장 별 DNA 분열을 위한 병렬 높은 처리량 단 분자 운동 분석

Site-specific DNA cleavage (SSDC) is a key step in many cellular processes, and it is crucial to gene editing. This work describes a kinetic assay capable ...

Real-Time Analysis of Bioenergetics in Primary Human Retinal Pigment Epithelial Cells Using High-Resolution Respirometry

Metabolic dysfunction of retinal pigment epithelial cells (RPE) is a key pathogenic driver of retinal diseases such as age-related macular degeneration (AMD) and proliferative vitreoretinopathy (PVR). Since RPE are highly metabolically-active cells, alterations in their metabolic status reflect changes in their health and function. High-resolution respirometry allows for real-time kinetic analysis of the two major bioenergetic pathways, glycolysis and mitochondrial oxidative phosphorylation (OXPHOS), through quantification of the extracellular acidification rate (ECAR) and oxygen consumption rate (OCR), respectively. The following is an optimized protocol for conducting high-resolution respirometry on primary human retinal pigment epithelial cells (H-RPE). This protocol provides a detailed description of the steps involved in producing bioenergetic profiles of RPE to define their basal and maximal OXPHOS and glycolytic capacities. Exposing H-RPE to different drug injections targeting the mitochondrial and glycolytic machinery results in defined bioenergetic profiles, from which key metabolic parameters can be calculated. This protocol highlights the enhanced response of BAM15 as an uncoupling agent compared to carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) to induce the maximal respiration capacity in RPE. This protocol can be utilized to study the bioenergetic status of RPE under different disease conditions and test the efficacy of novel drugs in restoring the basal metabolic status of RPE.

The metabolic status of human retinal pigment epithelial cells (H-RPE) reflects their health and function. Presented here is an optimized protocol for examining the real-time metabolic flux of H-RPE using high-resolution respirometry.

고분해능 호흡 측정법을 이용한 일차 인간 망막 색소 상피 세포의 생체 에너지 실시간 분석

Metabolic dysfunction of retinal pigment epithelial cells (RPE) is a key pathogenic driver of retinal diseases such as age-related macular degeneration ...

미토콘드리아 막 전위와 과산화물 수준의 정량화

Fluorescence-Based Quantification of Mitochondrial Membrane Potential and Superoxide Levels Using Live Imaging in HeLa Cells

Mitochondria are dynamic organelles critical for metabolic homeostasis by controlling energy production via ATP synthesis. To support cellular metabolism, various mitochondrial quality control mechanisms cooperate to maintain a healthy mitochondrial network. One such pathway is mitophagy, where PTEN-induced kinase 1 (PINK1) and Parkin phospho-ubiquitination of damaged mitochondria facilitate autophagosome sequestration and subsequent removal from the cell via lysosome fusion. Mitophagy is important for cellular homeostasis, and mutations in Parkin are linked to Parkinson's disease (PD). Due to these findings, there has been a significant emphasis on investigating mitochondrial damage and turnover to understand the molecular mechanisms and dynamics of mitochondrial quality control. Here, live-cell imaging was used to visualize the mitochondrial network of HeLa cells, to quantify the mitochondrial membrane potential and superoxide levels following treatment with carbonyl cyanide m-chlorophenyl hydrazone (CCCP), a mitochondrial uncoupling agent. In addition, a PD-linked mutation of Parkin (ParkinT240R) that inhibits Parkin-dependent mitophagy was expressed to determine how mutant expression impacts the mitochondrial network compared to cells expressing wild-type Parkin. The protocol outlined here describes a simple workflow using fluorescence-based approaches to quantify mitochondrial membrane potential and superoxide levels effectively.

저자 스포트라이트: HeLa 세포에서 실시간 이미징을 사용한 미토콘드리아 막 전위 및 과산화물 수준의 형광 기반 정량화  

This technique describes an effective workflow to visualize and quantitatively measure mitochondrial membrane potential and superoxide levels within HeLa cells using fluorescence-based live imaging.

HeLa 세포에서 라이브 이미징을 사용한 미토콘드리아 막 전위 및 슈퍼옥사이드 수준의 형광 기반 정량화

Mitochondria are dynamic organelles critical for metabolic homeostasis by controlling energy production via ATP synthesis. To support cellular metabolism, ...