Name: mitochondrial ca2+ retention capacity assay and ca2+-triggered mitochondrial swelling assay
Uploaded: 2018-05-01T12:30:00.000+00:00
Duration: 5 min 53 s
Description: Watch this Scientific Journal Video about Mitochondrial Ca2+ Retention Capacity Assay and Ca2+-triggered Mitochondrial Swelling Assay at JoVE.com

이 연구는 산동의 뛰어난 과학자 (JQ201421)와 NSFC (81371226)에서 부여의 부여에서 교부 금에 의해 지원 되었다.

1입니다. 미토 콘 드리 아의 격리
참고: 모든 솔루션 및 장비 0-4 ° C에 precooled와 있어야 얼음에 보관 합니다.
<ol><li>10 cm 세포 배양 접시 당 종자 약 3 x 106 SH SY5Y 셀. 높은 포도 당 Dulbecco에 문화 세포의 10% 태아 둔감 한 혈 청, 페니실린 (100 U/mL)와 글 중간을 수정-스 (100 µ g/mL). 밤새 5% CO2 를 포함 하는 인큐베이터에서 37 ° C에서 유지 합니다. 참고: 107 SH SY5Y 셀 x 적어도 1-2 각 격리 분석 결과 대 한 필요.</li>
	<li>매체를 제거 하 고 세척 약 1 mL 10 cm 세포 배양 접시에 얼음 차가운 PBS의 셀 3 번.</li>
	<li>10 cm 세포 배양 접시에 얼음 차가운 PBS의 새로운 1 mL을 추가 하 고 새로운 1.5 mL 튜브에 부착 세포를 긁어. 4 ° c.에서 10 분 동안 800 x g에서 원심 분리기</li>
	<li>원심 분리, 동안 5 mL 미토 콘 드 리아 절연 버퍼 (250 m m 자당, 10 mM HEPES (4-(2-hydroxyethyl)-1-piperazine ethanesulfonic 산), 10mm KCl, 1.5 m m MgCl2, 1 mM EDTA) protease 억제제 주식의 50 μ 보완 준비 솔루션 (100 x 농도; 1 EDTA 무료 태블릿 ddH2O의 500 µ L에 용 해).</li>
	<li>상쾌한을 제거 하 고 미토 콘 드리 아 절연 버퍼의 1 mL와 펠 릿을 resuspend. 30 분 동안 얼음에 1.5 mL 튜브를 품 어.</li>
	<li>Lyse 유리 균질 화기 (15-25 선)와 일시 중단 된 셀 아래로 수동으로 얼음에. 참고: 저기 없는 거품 세포 무 균 다는 것을 확인 하십시오. 현미경에 의해 그대로 셀과 드러난된 핵을 시각적으로 계산 하 고 확인 > 90% 셀 파손 발생 했습니다.</li>
	<li>1.5 mL 튜브 및 (핵 및 나머지 그대로 셀) 파편을 제거 하 4 ° C에서 5 분 동안 1000 x g에서 원심 분리기에는 homogenate를 전송 합니다.</li>
	<li>새로운 1.5 mL 튜브를 5 분 동안 1000 x g에서 원심 분리기에는 상쾌한 전송 4 ° c.</li>
	<li>새로운 튜브와 15 분 동안 14000 x g에서 원심 분리기에는 상쾌한 전송 4 ° c.</li>
	<li>상쾌한을 제거 하 고 미토 콘 드리 아 절연 버퍼의 1 mL와 함께 미토 콘 드리 아를 포함 하는 펠 릿을 resuspend.</li>
	<li>침전은 미토 콘 드리 아를 14000 x g 4 ° C에서 15 분에 대 한 솔루션을 원심.</li>
	<li>결과 미토 콘 드리 아 펠 릿 얼음에 보관 되어야 하며 50-100 μ KCl 미디어 (125 m KCl m, 2 m m K2HPO41 m m MgCl2, 20 mM HEPES, 5mm 조미료, 5mm malate, 및 2 μ M로 테 논, pH 7.4) 어떤 엉덩이 전에 펠 릿 볼륨에 따라 resuspended 불안.</li>
	<li>미토 콘 드 리아 솔루션의 5 μ를 사용 하 여 표준 BCA 분석 결과, OD562를 측정 하 여 미토 콘 드리 아 단백질 농도 측정 하는 플레이트 리더13를 사용 하 여.</li>
</ol>2. 미토 콘 드리 아 캘리포니아2 + 보존 용량 결정
<ol><li>KCl 미디어를 준비 하 고 추가 Ca2 +-실험 전에 0.5 μ M의 최종 농도에 녹색 형광 염료를 바인딩.</li>
	<li>고립 된 미토 콘 드리 아 (0.4 mg/mL)에 포함 된 0.5 μ M Ca2 +KCl 미디어의 1 mL를 전송-각 6 잘 플레이트에 녹색 형광 염료를 잘 바인딩.</li>
	<li>품 어는 미토 콘 드리 아 및 캘리포니아2 +-20 mM CaCl2 솔루션 (20 m m CaCl2, 127 m m KCl, 1 mM MgCl2 의 1 분 추가 4 μ aliquots에 대 한 주위 빛에서 보호 하는 실 온에서 6 잘 플레이트에서 녹색 형광 염료를 바인딩 20 mM HEPES, 5mm 조미료, 5mm malate, pH 7.4) 각 6-잘 접시에 잘 사용 하 여 자동 분배 200 nmol Ca2 +/mg 미토 콘 드리 아 단백질을 소개 하는 설정.</li>
	<li>모든 3 형광 변화를 모니터링 하는 형광 분석기 사용 506의 여기 파장을 2 분에 대 한 s 및 531의 방출 파장 nm. 접시 3 600 rpm에서 떨고 갖춘 소프트웨어 (그림 1)에 의해 제어 신호 사이 s.</li>
	<li>각 영역 20 mM CaCl2 솔루션의 추가 4 μ 약 수를 추가 하 고 모든 3 형광 변화를 모니터링 형광 시간 증가 될 때까지이 단계를 반복 하는 2 분에 대 한 s. 참고: 미토 콘 드리 아와 추가 Ca2 + 증가 기록 형광으로 표시도 전했다. mPTP가 열리면 형광 시간이 증가 합니다.</li>
	<li>캘리포니아2 + 는 mPTP Ca2 + 보존 용량으로 열릴 때까지 추가의 총 금액을 해석 합니다.</li>
</ol>3. 결정의 캘리포니아 2 +-미토 콘 드 리아 붓기를 유발
<ol><li>실험 전에 KCl 미디어를 준비 합니다.<ol>
<li>잘 각 6 잘 플레이트에 KCl 미디어에서 고립 된 미토 콘 드리 아 (0.4 mg/mL)의 1 mL를 전송.</li>
	</ol>
</li>
	<li>미토 콘 드 리아 붓기 하 미토 콘 드리 아 단백질으로 10 μ의 20mm CaCl2 수성 해결책 (500 nmol/mg 미토 콘 드리 아 단백질)를 추가 합니다.</li>
	<li>540에 흡 광도 감소를 기록 nm 매 3 10 분 (그림 2)에 대 한 소프트웨어에 의해 제어 microplate 리더에 s. 형광 변화 미토 콘 드리 아 내 막에 걸쳐 용액의 유입에 의해 발생 합니다. 참고: 540에서 감소 흡 광도 nm 부 미토 콘 드리 아에 해당. 독자 흡 광도 감소 관찰 되지 않는 경우는 더 긴 간격 읽거나 독서 시간을 채택 수 있습니다.</li>
</ol>

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X. 태양 실험을 감독. 웨이 리는 실험을 수행. 첸 장 및 X. 태양 종이 썼다.

여기, 우리는 미토 콘 드 리아 캘리포니아2 + 보존 용량 분석 결과 및 캘리포니아2 +에 대 한 간단 하 고 효과적인 프로토콜을 설명-미토 콘 드 리아 붓기 분석 결과 유발.
미토 콘 드리 아 격리에 대 한 모든 자료와 튜브는 얼음, 특히 때 세포 조직 확인 하십시오. 37 ° c.에 5 분 동안 접시에서 세포를 분리 하 0.25% trypsin EDTA는 사용할 수 있습니다. 후에 15-25 선, 그?...

미토 콘 드리 아 ATP 산화 인 산화에 의해 포유류 세포에서 사용의 거의 95%를 생산 주요 세포 기관 이다. 그것은 그의 캘리포니아2 + 미토 콘 드리 아에 의해 분리 된 micromolar 집중, ADP와 무기 인산 염의 존재를 사용할 수 있는 합성 ATP1ADP phosphorylate 보고. Cytosolic 캘리포니아2 + 의 농도 임계값 이상 되 면 미토 콘 드리 아 수 이해 캘리포니아2 + 급속 하 게 경과 하 고 그것은 천천히. 따라서, 작동 미토 콘 드리 아 수 유입 증가 cytosolic 캘리포니아2 +. 없는 산화 인 산화에 참여 하는 미토 콘 드 리아 Ca2 + 또한 cytosolic 칼슘 신호에 참여 및 미토 콘 드리 아 내에는 mPTP의 개방을 유도 하 여 미토 콘 드 리아 apoptotic 메커니즘을 활성화 막2. 그것은 널리 인정 되었습니다 세포내 캘리포니아2 + 의 비정상적인 상승 mPTP3여 미토 콘 드리 아 대규모 붓기를 일으킬 수 있다. 따라서,이 프로토콜 미토 콘 드 리아 캘리포니아2 + 보존 용량 및 캘리포니아2 +측정 하 여 미토 콘 드리 아 기능을 평가 하는 것을 목표로-유혹 trigged 미토 콘 드 리아 붓기.
캘리포니아2 + 보존 용량 글귀 cytosolic 칼슘을 미토 콘 드리 아의 능력의 측정입니다. 미토 콘 드리 아 무료 cytosolic 칼슘을 버퍼링 하 고 비활성 침전의 형태로 칼슘 통풍 관에 의해 칼슘-의존 세포질 과정을 조절 수 있습니다. 장애인된 캘리포니아2 + 보존 용량 에너지 제한 및 심지어 neurodegenerative 질병4,5와 관련 된 스트레스 현상에서 발생 합니다. 고립 된 미토 콘 드리 아 또는 셀 digitonin permeabilized Ca2 + 보존 용량, 식별 하는 데 수 고 축적 캘리포니아2 + 추가 조미료와 malate 고립 된 미토 콘 드리 아에 의해 상승 된 능력에 의해 영향을 받지는 합니다 미토 콘 드리 아 격리 절차6. 적정 한 양의 digitonin 플라즈마 막 종류의 다른 세포를 permeabilize를 사용 해야 합니다. 캘리포니아2 +의 hexapotassium 소금-녹색 형광 염료, 캘리포니아2 +바인딩-민감한 셀 impermeant 표시 빛 고르기 표시기, 널리7되었습니다. 캘리포니아2 +-(5 분) stimulations8연장 바인딩 녹색 형광 성 염료는 낮은 선호도 표시기 이며 무료 세포내 캘리포니아2 + 농도 동안 상승 계속 표시 하는 데 사용. 이 방법은 방사성 필터 기법 보다 덜 민감한 하지만 매우 간단 하 게 했다. 추가 Ca2 + 칼슘 녹색 형광을 끌어올리고 고 미토 콘 드리 아에 의해 Ca2 + 통풍 관 기준선에 형광을 반환 합니다. 캘리포니아2 + 의 순차적 추가 mitochondria 통풍 관 extramitochondrial Ca2 + 9실패까지 했다. 형광 microplate 리더는 지속적으로 칼슘 녹색 형광을 보고 사용할 수 있습니다.
캘리포니아2 + 축적, 후 미토 콘 드리 아 depolarized, 캘리포니아2 + , 매체에 발표 고 팽창 하기 시작 했다. 캘리포니아2 +-트리거 미토 콘 드리 아 붓기 mPTP 개방을 나타내는 데 사용 됩니다. 전자 현미경 및 측정 캘리포니아2 +데 540 nm 수에 빛 흡 광도 감소-미토 콘 드 리아 붓기10,11을 실행. 앞으로 각 산란12, 흡수도 감소 미토 콘 드 리아 매트릭스의 수동 붓기를 반영 미토 콘 드리 아 볼륨을 직접 확인할 수 있습니다.
여기, 우리는 미토 콘 드 리아 캘리포니아2 + 보존 용량 및 캘리포니아2 +검사 방법론 설명-SH-SY5Y 세포에서 고립 된 미토 콘 드리 아에서 미토 콘 드리 아 붓기를 트리거.

<table><tbody><tr><td>SH-SY5Y cell line</td><td>ATCC (The Global Bioresource Center)</td><td>ATCC Number: CRL-2266</td><td></td></tr><tr><td>Calcium green-5N</td><td>Life Technologies</td><td>c-3737</td><td>Ca&lt;sup&gt;2+&lt;/sup&gt;-binding green fluorescent dye</td></tr><tr><td>complete protease inhibitors</td><td>Roche Molecular Biochemicals</td><td>4693116001</td><td></td></tr><tr><td>glass homogenizer</td><td>Kimble Chase</td><td>9885303002</td><td></td></tr><tr><td>glutamate</td><td>Sigma-Aldrich</td><td>RES5063G-A7</td><td></td></tr><tr><td>malate</td><td>Sigma-Aldrich</td><td>46940-U</td><td></td></tr><tr><td>rotenone</td><td>Sigma-Aldrich</td><td>R8875</td><td></td></tr><tr><td>HEPES</td><td>Sigma-Aldrich</td><td>H3375</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>00457</td><td></td></tr><tr><td>K&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>V900050</td><td></td></tr><tr><td>KCl</td><td>Sigma-Aldrich</td><td>P9541</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>V900266</td><td></td></tr><tr><td>Varioskan flash instruments</td><td>Thermo Scientific</td><td>IC100E</td><td></td></tr><tr><td>Bongkrekate</td><td>Biovision</td><td>1820-100</td><td></td></tr><tr><td>atractyloside</td><td>Sigma-Aldrich</td><td>C4992</td><td></td></tr><tr><td>high-glucose Dulbecco&amp;rsquo;s modified Eagle medium</td><td>Hyclone</td><td>SH30022</td><td>4.5 g/L glucose, L-glutamine, without sodium pyruvate</td></tr><tr><td>fetal bovine serum</td><td>Hyclone</td><td>SV30087</td><td></td></tr><tr><td>penicillin</td><td>Sigma-Aldrich</td><td>P3032</td><td></td></tr><tr><td>streptomycin</td><td>Invitrogen</td><td>11860-038</td><td></td></tr><tr><td>BCA assay kit</td><td>Thermo Scientific</td><td>NCI3225CH</td><td></td></tr><tr><td>PBS</td><td>Hyclone</td><td>SH30256.01</td><td></td></tr><tr><td>10 cm cell culture dish</td><td>NEST</td><td>704001</td><td></td></tr></tbody></table>

mitochondrial ca2+ retention capacity assay and ca2+-triggered mitochondrial swelling assay

산화 인 산화에 의해 ATP의 생산은 미토 콘 드리 아의 주 기능은 이다. 더 높은 진핵생물의 미토 콘 드리 아도 참여할 cytosolic 캘리포니아2 + 버퍼링, 그리고 미토 콘 드리 아에서 ATP 생산 intramitochondrial 무료 캘리포니아2 + 농도 의해 중재 될 수 있다. 캘리포니아2 + 보존 용량 미토 콘 드 리아 매트릭스에 칼슘을 유지 하는 미토 콘 드리 아의 기능으로 간주 될 수 있습니다. 축적 된 세포내 캘리포니아2 + 리드 안 미토 콘 드리 아 막의 침투성을 미토 콘 드리 아 침투성 전환 기 공 (mPTP)는 분자와 분자의 유출에는 리드 무게 1.5 kDa의 개통 되 나. 캘리포니아2 +-트리거 미토 콘 드리 아 붓기 mPTP 개방을 나타내는 데 사용 됩니다. 여기, 우리가 설명 하는 Ca2 + 보존 용량 및 캘리포니아2 +검사를 두 분석-고립 된 미토 콘 드리 아에서 미토 콘 드리 아 붓기를 트리거. 특정 양의 캘리포니아2 + 추가 후 모든 단계는 하루에 완료 고 microplate 리더에 의해 기록 될 수 있습니다. 따라서,이 두 가지 간단 하 고 효과적인 분석 평가 Ca2 +채택 될 수-미토 콘 드 리아 기능 관련.

캘리포니아2 + 보존 용량의 대표 결과: 결과 형광 값으로 표현 되었다. 캘리포니아2 + (200 nmols/mg 미토 콘 드리 아 단백질)의 더블 mPTP 오프닝 베이스 위에 증가 하는 형광의 추가 펄스와. 5 μ M Bongkrekate (BKA), 캘리포니아2 +의 억제제-mPTP 개방, 또는 1 μ M atractyloside (ATR), 캘리포니아2 +의 활성 유도-mPTP 개방을 유도, 고립 된 미토 콘 드리 ...

Watch this Scientific Journal Video about Mitochondrial Ca2+ Retention Capacity Assay and Ca2+-triggered Mitochondrial Swelling Assay at JoVE.com

Mitochondrial Ca2+ Retention Capacity Assay and Ca2+-triggered Mitochondrial Swelling Assay

미토 콘 드 리아 캘리포니아2 + 보존 용량 분석 결과 및 캘리포니아2 +-미토 콘 드 리아 붓기 분석 결과 발생

이 프로토콜 Ca2 + 보존 용량 및 캘리포니아2 +검사 하는 방법을 설명 하는 것을 목표로-트리거 SH-SY5Y의 고립 된 미토 콘 드리 아의 붓기 미토 콘 드리 아 세포 단계.

The production of ATP by oxidative phosphorylation is the primary function of mitochondria. Mitochondria in higher eukaryotes also participate in ...

mitochondrial-ca2-retention-capacity-assay-ca2-triggered

Research

JoVE Journal

Neuroscience

Mitochondrial Ca2+ Retention Capacity Assay and Ca2+-triggered Mitochondrial Swelling Assay

11.1K 조회수.  Qilu Hospital of Shandong University. 이 프로토콜 Ca2 + 보존 용량 및 캘리포니아2 +검사 하는 방법을 설명 하는 것을 목표로-트리거 SH-SY5Y의 고립 된 미토 콘 드리 아의 붓기 미토 콘 드리 아 세포 단계.

비디오: Mitochondrial Ca2+ Retention Capacity Assay and Ca2+-triggered Mitochondrial Swelling Assay

Visualization of Mitochondrial DNA Replication in Individual Cells by EdU Signal Amplification

Mitochondria are key regulators of cellular energy and mitochondrial biogenesis is an essential component of regulating mitochondria numbers in healthy cells1-3. One approach for monitoring mitochondrial biogenesis is to measure the rate of mitochondrial DNA (mtDNA) replication4. We developed a sensitive technique to label newly synthesized mtDNA in individual cells in order to study mtDNA biogenesis. The technique combines the incorporation of 5-ethynyl-2'-deoxyuridine (EdU)5-7 with a tyramide signal amplification (TSA)8 protocol to visualize mtDNA replication within subcellular compartments of neurons. EdU is superior to other thymidine analogs, such as 5-bromo-2-deoxyuridine (BrdU), because the initial click reaction to label EdU5-7 does not require the harsh acid treatments or enzyme digests that are required for exposing the BrdU epitope. The milder labeling of EdU allows for direct comparison of its incorporation with other cellular markers9-10. The ability to visualize and quantify mtDNA biogenesis provides an essential tool for investigating the mechanisms used to regulate mitochondrial biogenesis and would provide insight into the pathogenesis associated with drug toxicity, aging, cancer and neurodegenerative diseases. Our technique is applicable to sensory neurons as well as other cell types. The use of this technique to measure mtDNA biogenesis has significant implications in furthering the understanding of both normal cellular physiology as well as impaired disease states.

We developed a sensitive technique to label newly synthesized mitochondrial DNA (mtDNA) in individual cells in order to study mtDNA biogenesis. The technique combines the incorporation of EdU together with a tyramide signal amplification (TSA) protocol to visualize mtDNA replication within subcellular compartments of neurons.

에듀 신호 증폭 개별 세포 Mitochondrial DNA 복제의 시각화

Mitochondria are key regulators of cellular energy and mitochondrial biogenesis is an essential component of regulating mitochondria numbers in healthy ...

연구

신경과학

Neuromodulation and Mitochondrial Transport: Live Imaging in Hippocampal Neurons over Long Durations

To understand the relationship between mitochondrial transport and neuronal function, it is critical to observe mitochondrial behavior in live cultured neurons for extended durations1-3. This is now possible through the use of vital dyes and fluorescent proteins with which cytoskeletal components, organelles, and other structures in living cells can be labeled and then visualized via dynamic fluorescence microscopy. For example, in embryonic chicken sympathetic neurons, mitochondrial movement was characterized using the vital dye rhodamine 1234. In another study, mitochondria were visualized in rat forebrain neurons by transfection of mitochondrially targeted eYFP5. However, imaging of primary neurons over minutes, hours, or even days presents a number of issues. Foremost among these are: 1) maintenance of culture conditions such as temperature, humidity, and pH during long imaging sessions; 2) a strong, stable fluorescent signal to assure both the quality of acquired images and accurate measurement of signal intensity during image analysis; and 3) limiting exposure times during image acquisition to minimize photobleaching and avoid phototoxicity.
Here, we describe a protocol that permits the observation, visualization, and analysis of mitochondrial movement in cultured hippocampal neurons with high temporal resolution and under optimal life support conditions. We have constructed an affordable stage-top incubator that provides good temperature regulation and atmospheric gas flow, and also limits the degree of media evaporation, assuring stable pH and osmolarity. This incubator is connected, via inlet and outlet hoses, to a standard tissue culture incubator, which provides constant humidity levels and an atmosphere of 5-10% CO2/air. This design offers a cost-effective alternative to significantly more expensive microscope incubators that don't necessarily assure the viability of cells over many hours or even days. To visualize mitochondria, we infect cells with a lentivirus encoding a red fluorescent protein that is targeted to the mitochondrion. This assures a strong and persistent signal, which, in conjunction with the use of a stable xenon light source, allows us to limit exposure times during image acquisition and all but precludes photobleaching and phototoxicity. Two injection ports on the top of the stage-top incubator allow the acute administration of neurotransmitters and other reagents intended to modulate mitochondrial movement. In sum, lentivirus-mediated expression of an organelle-targeted red fluorescent protein and the combination of our stage-top incubator, a conventional inverted fluorescence microscope, CCD camera, and xenon light source allow us to acquire time-lapse images of mitochondrial transport in living neurons over longer durations than those possible in studies deploying conventional vital dyes and off-the-shelf life support systems.

We describe a protocol that allows imaging of mitochondria in living neurons via fluorescence microscopy over long durations. Imaging over extended periods is accomplished through lentivirus-mediated expression of a mitochondrially targeted fluorescent protein and use of an inexpensive stage-top incubator that was designed and built in our laboratory.

Neuromodulation 및 Mitochondrial 교통 : 긴 기간이 이상 Hippocampal 뉴런의 라이브 영상

To understand the relationship between mitochondrial transport and neuronal function, it is critical to observe mitochondrial behavior in live cultured ...

Determination of Mitochondrial Membrane Potential and Reactive Oxygen Species in Live Rat Cortical Neurons

Mitochondrial membrane potential (&Delta;&Psi;m) is critical for maintaining the physiological function of the respiratory chain to generate ATP. A significant loss of &Delta;&Psi;m renders cells depleted of energy with subsequent death. Reactive oxygen species (ROS) are important signaling molecules, but their accumulation in pathological conditions leads to oxidative stress. The two major sources of ROS in cells are environmental toxins and the process of oxidative phosphorylation. Mitochondrial dysfunction and oxidative stress have been implicated in the pathophysiology of many diseases; therefore, the ability to determine &Delta;&Psi;m and ROS can provide important clues about the physiological status of the cell and the function of the mitochondria. 
Several fluorescent probes (Rhodamine 123, TMRM, TMRE, JC-1) can be used to determine &Delta;&psi;m in a variety of cell types, and many fluorescence indicators (Dihydroethidium, Dihydrorhodamine 123, H2DCF-DA) can be used to determine ROS. Nearly all of the available fluorescence probes used to assess &Delta;&Psi;m or ROS are single-wavelength indicators, which increase or decrease their fluorescence intensity proportional to a stimulus that increases or decreases the levels of &Delta;&Psi;m or ROS. Thus, it is imperative to measure the fluorescence intensity of these probes at the baseline level and after the application of a specific stimulus. This allows one to determine the percentage of change in fluorescence intensity between the baseline level and a stimulus. This change in fluorescence intensity reflects the change in relative levels of &Delta;&Psi;m or ROS. In this video, we demonstrate how to apply the fluorescence indicator, TMRM, in rat cortical neurons to determine the percentage change in TMRM fluorescence intensity between the baseline level and after applying FCCP, a mitochondrial uncoupler. The lower levels of TMRM fluorescence resulting from FCCP treatment reflect the depolarization of mitochondrial membrane potential. We also show how to apply the fluorescence probe H2DCF-DA to assess the level of ROS in cortical neurons, first at baseline and then after application of H2O2. This protocol (with minor modifications) can be also used to determine changes in &#x2206;&#x03A8;m and ROS in different cell types and in neurons isolated from other brain regions.

We demonstrate application of the fluorescence indicator, TMRM, in cortical neurons to determine the relative changes in TMRM fluorescence intensity before and after application of a specific stimulus. We also show application of the fluorescence probe H2DCF-DA to assess the relative level of reactive oxygen species in cortical neurons.

라이브 랫 두피 뉴런에서 Mitochondrial 분리막 잠재력과 반응 산소 종의 결정

Mitochondrial membrane potential (&Delta;&Psi;m) is critical for maintaining the physiological function of the respiratory chain to generate ATP.  A ...

Analyses of Mitochondrial Calcium Influx in Isolated Mitochondria and Cultured Cells

Ca2+ handling by mitochondria is a critical function regulating both physiological and pathophysiological processes in a broad spectrum of cells. The ability to accurately measure the influx and efflux of Ca2+ from mitochondria is important for determining the role of mitochondrial Ca2+ handling in these processes. In this report, we present two methods for the measurement of mitochondrial Ca2+ handling in both isolated mitochondria and cultured cells. We first detail a plate reader-based platform for measuring mitochondrial Ca2+ uptake using the Ca2+ sensitive dye calcium green-5N. The plate reader-based format circumvents the need for specialized equipment, and the calcium green-5N dye is ideally suited for measuring Ca2+ from isolated tissue mitochondria. For our application, we describe the measurement of mitochondrial Ca2+ uptake in mitochondria isolated from mouse heart tissue; however, this procedure can be applied to measure mitochondrial Ca2+ uptake in mitochondria isolated from other tissues such as liver, skeletal muscle, and brain. Secondly, we describe a confocal microscopy-based assay for measurement of mitochondrial Ca2+ in permeabilized cells using the Ca2+ sensitive dye Rhod-2/AM and imaging using 2-dimensional laser-scanning microscopy. This permeabilization protocol eliminates cytosolic dye contamination, allowing for specific recording of changes in mitochondrial Ca2+. Moreover, laser-scanning microscopy allows for high frame rates to capture rapid changes in mitochondrial Ca2+ in response to various drugs or reagents applied in the external solution. This protocol can be applied to measure mitochondrial Ca2+ uptake in many cell types including primary cells such as cardiac myocytes and neurons, and immortalized cell lines.

Here, we present two protocols for the measurement of mitochondrial Ca2+ influx in isolated mitochondria and cultured cells. For isolated mitochondria, we detail a plate reader-based Ca2+ import assay using the Ca2+ sensitive dye calcium green-5N. For cultured cells, we describe a confocal microscopy method using the Ca2+ dye Rhod-2/AM.

고립 된 미토 콘 드리 아 및 경작된 한 세포에 있는 미토 콘 드 리아 칼슘 유입의 분석

Ca2+ handling by mitochondria is a critical function regulating both physiological and pathophysiological processes in a broad spectrum of cells. The ...

생화학

Monitoring Dynamic Changes In Mitochondrial Calcium Levels During Apoptosis Using A Genetically Encoded Calcium Sensor

Dynamic changes in intracellular calcium concentration in response to various stimuli regulates many cellular processes such as proliferation, differentiation, and apoptosis1. During apoptosis, calcium accumulation in mitochondria promotes the release of pro-apoptotic factors from the mitochondria into the cytosol2. It is therefore of interest to directly measure mitochondrial calcium in living cells in situ during apoptosis. High-resolution fluorescent imaging of cells loaded with dual-excitation ratiometric and non-ratiometric synthetic calcium indicator dyes has been proven to be a reliable and versatile tool to study various aspects of intracellular calcium signaling. Measuring cytosolic calcium fluxes using these techniques is relatively straightforward. However, measuring intramitochondrial calcium levels in intact cells using synthetic calcium indicators such as rhod-2 and rhod-FF is more challenging. Synthetic indicators targeted to mitochondria have blunted responses to repetitive increases in mitochondrial calcium, and disrupt mitochondrial morphology3. Additionally, synthetic indicators tend to leak out of mitochondria over several hours which makes them unsuitable for long-term experiments. Thus, genetically encoded calcium indicators based upon green fluorescent protein (GFP)4 or aequorin5 targeted to mitochondria have greatly facilitated measurement of mitochondrial calcium dynamics. Here, we describe a simple method for real-time measurement of mitochondrial calcium fluxes in response to different stimuli. The method is based on fluorescence microscopy of 'ratiometric-pericam' which is selectively targeted to mitochondria. Ratiometric pericam is a calcium indicator based on a fusion of circularly permuted yellow fluorescent protein and calmodulin4. Binding of calcium to ratiometric pericam causes a shift of its excitation peak from 415 nm to 494 nm, while the emission spectrum, which peaks around 515 nm, remains unchanged. Ratiometric pericam binds a single calcium ion with a dissociation constant in vitro of ~1.7 &mu;M4. These properties of ratiometric pericam allow the quantification of rapid and long-term changes in mitochondrial calcium concentration. Furthermore, we describe adaptation of this methodology to a standard wide-field calcium imaging microscope with commonly available filter sets. Using two distinct agonists, the purinergic agonist ATP and apoptosis-inducing drug staurosporine, we demonstrate that this method is appropriate for monitoring changes in mitochondrial calcium concentration with a temporal resolution of seconds to hours. Furthermore, we also demonstrate that ratiometric pericam is also useful for measuring mitochondrial fission/fragmentation during apoptosis. Thus, ratiometric pericam is particularly well suited for continuous long-term measurement of mitochondrial calcium dynamics during apoptosis.

This protocol describes a method for real-time measurement of mitochondrial calcium fluxes by fluorescent imaging. The method takes advantage of a circularly permutated YFP-based dual-excitation ratiometric calcium sensor (ratiometric pericam-mt) selectively expressed in mitochondria.

유전자 변형으로 인코딩된 칼슘 센서를 사용하여 Apoptosis 동안 Mitochondrial 칼슘 레벨에서 동적 변경 사항 모니터링

Dynamic changes in intracellular calcium concentration in response to various stimuli regulates many cellular processes such as proliferation, ...

생물학

Multi-parameter Measurement of the Permeability Transition Pore Opening in Isolated Mouse Heart Mitochondria

The mitochondrial permeability transition pore (mtPTP) is a non specific channel that forms in the inner mitochondrial membrane to transport solutes with a molecular mass smaller than 1.5 kDa. Although the definitive molecular identity of the pore is still under debate, proteins such as cyclophilin D, VDAC and ANT contribute to mtPTP formation. While the involvement of mtPTP opening in cell death is well established1, accumulating evidence indicates that the mtPTP serves a physiologic role during mitochondrial Ca2+ homeostasis2, bioenergetics and redox signaling 3.
mtPTP opening is triggered by matrix Ca2+ but its activity can be modulated by several other factors such as oxidative stress, adenine nucleotide depletion, high concentrations of Pi, mitochondrial membrane depolarization or uncoupling, and long chain fatty acids4. In vitro, mtPTP opening can be achieved by increasing Ca2+ concentration inside the mitochondrial matrix through exogenous additions of Ca2+ (calcium retention capacity). When Ca2+ levels inside mitochondria reach a certain threshold, the mtPTP opens and facilitates Ca2+ release, dissipation of the proton motive force, membrane potential collapse and an increase in mitochondrial matrix volume (swelling) that ultimately leads to the rupture of the outer mitochondrial membrane and irreversible loss of organelle function.
Here we describe a fluorometric assay that allows for a comprehensive characterization of mtPTP opening in isolated mouse heart mitochondria. The assay involves the simultaneous measurement of 3 mitochondrial parameters that are altered when mtPTP opening occurs: mitochondrial Ca2+ handling (uptake and release, as measured by Ca2+ concentration in the assay medium), mitochondrial membrane potential, and mitochondrial volume. The dyes employed for Ca2+ measurement in the assay medium and mitochondrial membrane potential are Fura FF, a membrane impermeant, ratiometric indicator which undergoes a shift in the excitation wavelength in the presence of Ca2+, and JC-1, a cationic, ratiometric indicator which forms green monomers or red aggregates at low and high membrane potential, respectively. Changes in mitochondrial volume are measured by recording light scattering by the mitochondrial suspension. Since high-quality, functional mitochondria are required for the mtPTP opening assay, we also describe the steps necessary to obtain intact, highly coupled and functional isolated heart mitochondria.

A spectrofluorometric protocol for the measurement of the mitochondrial permeability transition pore opening in isolated mouse heart mitochondria is presented here. The assay involves the simultaneous measurement of mitochondria Ca2+ handling, mitochondrial membrane potential and mitochondrial volume. The procedure for obtaining high-quality and functional heart mitochondria is also described.

미토콘드리아 절연 마우스 중심부에 투과율 전환 기공 영업의 다중 매개 변수 측정

The mitochondrial permeability transition pore (mtPTP) is a non specific channel that forms in the inner mitochondrial membrane to transport solutes with ...

Simultaneous Measurement of Mitochondrial Calcium and Mitochondrial Membrane Potential in Live Cells by Fluorescent Microscopy

Apart from their essential role in generating ATP, mitochondria also act as local calcium (Ca2+) buffers to tightly regulate intracellular Ca2+ concentration. To do this, mitochondria utilize the electrochemical potential across their inner membrane (&#916;&#936;m) to sequester Ca2+. The influx of Ca2+ into the mitochondria stimulates three rate-limiting dehydrogenases of the citric acid cycle, increasing electron transfer through the oxidative phosphorylation (OXPHOS) complexes. This stimulation maintains &#916;&#936;m, which is temporarily dissipated as the positive calcium ions cross the mitochondrial inner membrane into the mitochondrial matrix.
We describe here a method for simultaneously measuring mitochondria Ca2+ uptake and &#916;&#936;m in live cells using confocal microscopy. By permeabilizing the cells, mitochondrial Ca2+ can be measured using the fluorescent Ca2+ indicator Fluo-4, AM, with measurement of &#916;&#936;m using the fluorescent dye tetramethylrhodamine, methyl ester, perchlorate (TMRM). The benefit of this system is that there is very little spectral overlap between the fluorescent dyes, allowing accurate measurement of mitochondrial Ca2+ and &#916;&#936;m simultaneously. Using the sequential addition of Ca2+ aliquots, mitochondrial Ca2+ uptake can be monitored, and the concentration at which Ca2+ induces mitochondrial membrane permeability transition and the loss of &#916;&#936;m determined.

Mitochondria can utilize the electrochemical potential across their inner membrane (&#916;&#936;m) to sequester calcium (Ca2+), allowing them to shape cytosolic Ca2+ signaling within the cell. We describe a method for simultaneously measuring mitochondria Ca2+ uptake and &#916;&#936;m in live cells using fluorescent dyes and confocal microscopy.

형광 현미경으로 살아있는 세포의 잠재적 인 미토콘드리아의 칼슘과 미토콘드리아 막의 동시 측정

Apart from their essential role in generating ATP, mitochondria also act as local calcium (Ca2+) buffers to tightly regulate intracellular Ca2+ ...

Calcium Influx Assay to Measure Mitochondrial Calcium Uptake in Cultured Cells

Source: Maxwell, J.T., et al., <a href="https://app.jove.com/v/57225">Analyses of Mitochondrial Calcium Influx in Isolated Mitochondria and Cultured Cells.</a> J. Vis. Exp. (2018).
This video describes a confocal microscopy imaging-based assay to measure mitochondrial calcium uptake in cultured cells. The assay uses the calcium-sensitive fluorescent dye Rhod-2/AM to measure the mitochondrial calcium in permeabilized cultured cells.

배양된 세포에서 미토콘드리아 칼슘 흡수를 측정하기 위한 칼슘 유입 분석

Source: Maxwell, J.T., et al., Analyses of Mitochondrial Calcium Influx in Isolated Mitochondria and Cultured Cells. J. Vis. Exp. (2018).
This video ...

JoVE Encyclopedia of Experiments

Biological Techniques

Assay Techniques

Cell-based assays

Mitochondrial Calcium Uptake Assay: A Plate Reader-Based Method for Measuring Calcium Uptake in Isolated Mitochondria

Source: Maxwell, J.T., et al., <a href="https://app.jove.com/v/57225">Analyses of Mitochondrial Calcium Influx in Isolated Mitochondria and Cultured Cells.</a> J. Vis. Exp. (2018).
This video describes a plate reader-based assay to measure mitochondrial calcium uptake in isolated mitochondria. The assay analyzes the fluorescence signal of a calcium-sensitive fluorescent dye to study the kinetics of mitochondrial calcium uptake and calcium overload.

미토콘드리아 칼슘 흡수 분석법: 분리된 미토콘드리아에서 칼슘 흡수를 측정하기 위한 플레이트 리더 기반 방법

Biochemical assays

Determination of the Calcium Retention Capacity of Isolated Mitochondria

Source: Li, W., et. al. <a href="https://app.jove.com/v/56236">Mitochondrial Ca2+ Retention Capacity Assay and Ca2+-triggered Mitochondrial Swelling Assay.</a> J. Vis. Exp. (2018)
This video demonstrates the procedure to assess mitochondrial calcium retention capacity by monitoring real-time changes in fluorescence as mitochondria uptake calcium. This assay is useful for studying mitochondrial function and calcium-related processes, including apoptosis and neurodegenerative diseases.

Source: Li, W., et. al. Mitochondrial Ca2+ Retention Capacity Assay and Ca2+-triggered Mitochondrial Swelling Assay. J. Vis. Exp. (2018)
This video ...

미토 콘 드 리아 캘리포니아^{2 +} 보존 용량 분석 결과 및 캘리포니아^{2 +}-미토 콘 드 리아 붓기 분석 결과 발생

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