이 작품은 국립 보건원, 국립 당뇨병 연구소와 소화기 및 신장병, 2R01DK59558 (GN까지)에서 보조금에 의해 지원되었다. 연구 자료 보조금 UL1 RR029884을위한 건강 국립 센터의 국립 연구소에 의해 지원 UAMS 병진 연구소 UAMS에서 유동 세포 계측법 코어의 일부 자금을 제공했습니다. 우리가 고마워 박사 Peipei 핑 (로스 앤젤레스 캘리포니아 대학, 로스 앤젤레스, CA) cDNA PKC-ε 박사 알렌 Samarel (로욜라 메리 대학 의료 센터의 지배적 부정적 (비활성) 돌연변이 코딩 휴대 아데노 바이러스의 나누어지는를 제공, PKC-ε의 constitutively 활성화 돌연변이 코딩 adenoviral 벡터의 나누어지는를 제공 Maywood, IL). 우리는 또한 Drs 감사드립니다. PKC-ε constitutively 활성화 cDNA 코딩을 제공하는 피터 파커와 피터 Sugden (임페리얼 칼리지 런던, 런던, 영국).

<ol>
	<li>Nowak, G., Schnellmann, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+culture+conditions+stimulate+gluconeogenesis+in+primary+cultures+of+renal+proximal+tubule+cells.">Improved culture conditions stimulate gluconeogenesis in primary cultures of renal proximal tubule cells.</a> Am. J. Physiol. 268, C1053-C1061 (1995).</li><li>Nowak, G., Schnellmann, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=L-ascorbic+acid+regulates+growth+and+metabolism+of+renal+cells:+improvements+in+cell+culture.">L-ascorbic acid regulates growth and metabolism of renal cells: improvements in cell culture.</a> Am. J. Physiol. 271, 2072-2080 (1996).</li><li>Ping, P., Takano, H., Zhang, J., Tang, X. L., Qiu, Y., Li, R. C., Banerjee, S., Dawn, B., Balafonova, Z., Bolli, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isoform-selective+activation+of+protein+kinase+C+by+nitric+oxide+in+the+heart+of+conscious+rabbits:+a+signaling+mechanism+for+both+nitric+oxide-induced+and+ischemia-induced+preconditioning.">Isoform-selective activation of protein kinase C by nitric oxide in the heart of conscious rabbits: a signaling mechanism for both nitric oxide-induced and ischemia-induced preconditioning.</a> Circ. Res. 84, 587-604 (1999).</li><li>Wotton, D., Ways, D. K., Parker, P. J., Owen, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activity+of+both+Raf+and+Ras+is+necessary+for+activation+of+transcription+of+the+human+T+cell+receptor+beta+gene+by+protein+kinase+C,+Ras+plays+multiple+roles.">Activity of both Raf and Ras is necessary for activation of transcription of the human T cell receptor beta gene by protein kinase C, Ras plays multiple roles.</a> J. Biol. Chem. 268, 17975-17982 (1993).</li><li>Nowak, G., Bakajsova, D., Samarel, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+kinase+C-&#949;+activation+induces+mitochondrial+dysfunction+and+fragmentation+in+renal+proximal+tubules.">Protein kinase C-&#949; activation induces mitochondrial dysfunction and fragmentation in renal proximal tubules.</a> Am. J. Physiol. Renal Physiol. 301, F197-F208 (2011).</li><li>Nowak, G., Clifton, G. L., Godwin, M. L., Bakajsova, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+ERK1/2+pathway+mediates+oxidant-induced+decreases+in+mitochondrial+function+in+renal+cells.">Activation of ERK1/2 pathway mediates oxidant-induced decreases in mitochondrial function in renal cells.</a> Am. J. Physiol. Renal Physiol. 291, 840-855 (2006).</li><li>Shaik, Z. P., Fifer, E. K., Nowak, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Akt+activation+improves+oxidative+phosphorylation+in+renal+proximal+tubular+cells+following+nephrotoxicant+injury.">Akt activation improves oxidative phosphorylation in renal proximal tubular cells following nephrotoxicant injury.</a> Am. J. Physiol. Renal Physiol. 294, F423-F432 (2008).</li><li>Nowak, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+kinase+C-&#945;+and+ERK1/2+mediate+mitochondrial+dysfunction,+decreases+in+active+Na++transport,+and+cisplatin-induced+apoptosis+in+renal+cells.">Protein kinase C-&#945; and ERK1/2 mediate mitochondrial dysfunction, decreases in active Na+ transport, and cisplatin-induced apoptosis in renal cells.</a> J. Biol. Chem. 277, 43377-43388 (2002).</li><li>Liu, X., Godwin, M. L., Nowak, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+kinase+C-+a+inhibits+the+repair+of+oxidative+phosphorylation+after+S-(1,2-dichlorovinyl)-L-cysteine+injury+in+renal+cells.">Protein kinase C- a inhibits the repair of oxidative phosphorylation after S-(1,2-dichlorovinyl)-L-cysteine injury in renal cells.</a> Am. J. Physiol. Renal Physiol. 287, F64-F73 (2004).</li><li>Nowak, G., Price, P. M., Schnellmann, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lack+of+a+functional+p21WAF1/CIP1+gene+accelerates+caspase-independent+apoptosis+induced+by+cisplatin+in+renal+cells.">Lack of a functional p21WAF1/CIP1 gene accelerates caspase-independent apoptosis induced by cisplatin in renal cells.</a> Am. J. Physiol. Renal Physiol. 285, F440-F450 (2003).</li><li>Cummings, B. S., Schnellmann, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cisplatin-induced+renal+cell+apoptosis:+caspase+3-dependent+and+-independent+pathways.">Cisplatin-induced renal cell apoptosis: caspase 3-dependent and -independent pathways.</a> J. Pharmacol. Exp. Ther. 302, 8-17 (2002).</li></ol>

관심 없음 충돌이 선언 없습니다.

여기에 제시된 방법은 신장 근위 관 세포의 주요 문화에 PKC의 개별 isozymes의 overexpression 할 수 있습니다. 이 방법의 몇 가지 장점이 있습니다 : 1. 그것은 다양한 모욕 (국소 빈혈, hypoxia, 산화 스트레스), 약물, 그리고 신장에서 nephrotoxicants의 기본 목표 아르 셀 (신장 근위 관 세포)의 동질적인 인구 규제 메커니즘을 조사 할 수 있습니다. 2. 개선 된 조건에서 차 문화에서 자란 RPTC의 체외 모델에서?...

<table border="1"><tbody><tr><td> 시약의 이름 </td><td> 회사 </td><td> 카탈로그 번호 </td></tr><tr><td> 층류 흐름 후드 </td><td> 열 전자 주식회사 </td><td> 견적 1,104 </td></tr><tr><td> 2 ML 15 ML의 다운스 호 모지 조직 연삭기 </td><td> 휘튼 </td><td> 989-24607, 357,544 </td></tr><tr><td> 85 235 마이크론 나일론 메쉬 </td><td> 소형 부품 </td><td> CMN - 0085 - 10YD CMN - 0250 - 10YD </td></tr><tr><td> 50 ML 멸균 원심 분리기 관 </td><td> BIOLOGIX </td><td> BCT-P 50BS </td></tr><tr><td> 1.5 ML 마이크로 튜브 </td><td> Sarstedt </td><td> 72.690.001 </td></tr><tr><td> 35 X 10mm 멸균 문화 요리 </td><td> 코닝 </td><td> 430165 </td></tr><tr><td> 주앙의 원심 분리기 </td><td> 주앙 </td><td> 주앙 CR3 11,175,704 회 전자 : 주앙 T40 </td></tr><tr&gt; <td> 조절 마이크로 원심 분리기 </td><td> 시그마 </td><td> 모델 1-15 </td></tr><tr><td> 생물 산소 모니터 </td><td> 어떡하지 주식회사 </td><td> 어떡 모델 5300A </td></tr><tr><td> 싱글 상공 회의소 마이크로 산소 시스템 </td><td> 어떡하지 주식회사 </td><td> 5356S </td></tr><tr><td> 산소 프로브 </td><td> 어떡하지 주식회사 </td><td> 5331A </td></tr><tr><td> 목욕을 순환 </td><td> 어떡하지 주식회사 </td><td> 5310 </td></tr><tr><td> KCl과 표준 막 키트 </td><td> 어떡하지 주식회사 </td><td> 5775 </td></tr><tr><td> 자기 교반기 </td><td> 어떡하지 주식회사 </td><td> 5222 </td></tr><tr><td> 평판 레코더 </td><td> Kipp &amp; Zonen </td><td> BD 11E </td></tr><tr><td> 48 우물과 96도 투명 판 </td><td> Costar </td><td> 3548, 3679 </td></tr><tr><td> Thermomixer R </td><td> Eppendorf </td><td> 5355 21919 </td></tr><tr><td> 궤도 흔드는 MAXQ 2000 </td><td> 열 과학 </td><td> SHKA 2000 </td></tr><tr><td> 스펙트럼 형석 플러스 (흡광도 / 형광 / 발광 리더) </td><td> Tecan </td><td> F129005 </td></tr><tr><td> 물 외피 미국 Autoflow 자동 CO 2 인큐베이터 </td><td> NUAIRE </td><td> NU 4850 </td></tr><tr><td> 유동 세포 계측법에 대한 12x75 mm의 폴리스티렌 문화 테스트 튜브 </td><td> 피셔 브랜드 </td><td> 14-961-20 </td></tr><tr><td> Axioskop 물 침지 목표 63x / 0,90 W </td><td> 칼 Zeiss </td><td> 114846 ACHROPLAN 44 00 67 </td></tr><tr><td> DMEM / F12 </td><td> Cellgro </td><td> 99-830 - PB </td></tr><tr><td> DMEM / F12 햄 </td><td> 시그마 </td><td> D 2906 - 1L </td></tr><tr><td> Deferoxamine의 Mesylate </td><td> HospiRA </td><td> D110 </td></tr><tr><td> Collagenase 유형 I </td><td> 워싱턴 </td><td> 4196 </td></tr><tr><td> 트립신 억제제 </td><td> 시그마 </td><td> T 6522 - 500mg </td></tr><tr><td> 5,5 &#39;, 6,6&#39;- tetrachloro-1, 1 &#39;, 3,3&#39;이 - tetraethylbenzimidazolylcarbocyanine 요오드화물 (JC-1) </td><td> Invitrogen </td><td> T3168 </td></tr><tr><td> Mitotracker 레드 </td><td> Invitrogen </td><td> M22425 </td></tr><tr><td> ATP Bioluminescence 검정 키트 HS II </td><td> 로슈 </td><td> 11 699 709 001 </td></tr><tr><td> Annexin V - FITC 솔루션 </td><td> BioVision </td><td> 1001-200 </td></tr><tr><td> 흐름 cytometer </td><td> BD Biosciences </td><td> BD FACSCalibur </td></tr></tbody></table>

assessment of mitochondrial functions and cell viability in renal cells overexpressing protein kinase c isozymes

isozymes의 단백질 키나제 C (PKC) 가족 여러 생리 학적 및 병리학 과정에 참여하고 있습니다. 최근 데이터는 PKC는 mitochondrial 기능과 세포의 에너지 상태를 조절하는 보여줍니다. 많은 보고서는 PKC-a와 PKC-ε의 활성화는 허혈성 심장과 mediates의 cardioprotection에서 mitochondrial 기능을 향상 보여 주었다. 반면에, 우리는 PKC-α와 PKC-ε은 신장 세포에서 nephrotoxicant 유발 mitochondrial 장애 및 세포 죽음에 연루되어 보여주고 있습니다. 따라서, 본 연구의 목표는 PKC의 isozymes가 선택적으로 활성화 또는 산화 인산화와 세포 생존의 규정에서 자신의 역할을 결정하기 위해 저해 할 수있는 활성 mitochondrial 기능을 유지하는 신장 세포의 체외 모델을 개발하는 것이 었습니다. 신장 근위 관 세포 (RPTC)의 기본 문화는 미토의 mitochondrial 호흡과 활동의 결과로 개선 된 조건에서 배양 된생체 내 RPTC에서 비슷한 chondrial 효소. 전통 transfection 기술 (Lipofectamine, electroporation)가 기본 문화에 비효율적이며, mitochondrial 기능에 부정적 영향을 미칠 때문에, PKC-ε 돌연변이 cDNAs가 adenoviral 벡터를 통해 RPTC에게 전달했다. % 90 교양 RPTC의 transfection에있는이 방법은 결과를 표시합니다. 여기, 우리는에서 PKC-ε의 역할을 평가하기위한 방법을 제시 1. mitochondrial 형태와 ATP 합성, 2와 관련된 기능을 규제. 차 문화에 RPTC의 생존. PKC-ε는 overexpressing constitutively 활성화 PKC-ε 돌연변이에 의해 활성화됩니다. PKC-ε는 overexpressing에 의해 PKC-ε의 비활성 돌연변이를 억제하고 있습니다. Mitochondrial 기능, 호흡 단지 및 활동을 호흡기 체인의 무결성을 호흡을 검토하여 평가된다 F 0 F 1 ATPase, ATP 생산 속도, 및 ATP의 콘텐츠입니다. 호흡은주 3 일 (초과 기판과 ADP의 존재의 최대 호흡)와 uncoupled 호흡으로 digitonin-permeabilized RPTC에 ssessed. 호흡기 체인의 무결성은 절연 된 미토콘드리아의 호흡 체인의 네 가지 단지의 활동을 측정하여 평가된다. 산화 인산화의 용량은 F 0 F 1 ATPase의 ATP 생산 속도, 활동, mitochondrial 막 잠재력을 측정하여 평가됩니다. RPTC의 에너지 상태가 세포 내 ATP 내용을 결정함으로써 평가된다. 라이브 셀의 Mitochondrial 형태는 MitoTracker 레드 580, 특히 미토콘드리아에 축적 형광 염료, 라이브 monolayers 형광 현미경으로 검사 아르를 사용하여 시각화합니다. RPTC 가능성은 annexin V / apoptosis 및 oncosis을 결정하는 유동 세포 계측법에 이어 propidium의 요오드화물 착색를 사용하여 평가된다. 이러한 방법은 각각의 PKC isozymes의 선택적 활성화 / 억제 할 수 있도록체외의에 복제 할 수 있습니다 생리학 및 병리학 조건 다양한 세포 기능에서 자신의 역할을 평가합니다.

그림 1 프로그램 그 adenoviral 전달 RPTC와 미토콘드리아에서 PKC-ε의 크게 증가 단백질 수준에서 PKC-ε 결과의 constitutively 활성 (caPKC-ε)과 비활성 (dnPKC-ε) 뮤턴트 코딩 cDNA. 세포가 비활성화했습니다 cDNA 코딩 dnPKC을 ε overexpressed PKC-ε (phosphorylated되지 않음) (그림 1)에 감염된 반면 caPKC-ε 벡터를 들고 cDNA에 감염된 세포는 PKC-ε의 phosphorylated (활성) 양식을 overexpressed. 활성의 존재...

Watch this Scientific Journal Video about Assessment of Mitochondrial Functions and Cell Viability in Renal Cells Overexpressing Protein Kinase C Isozymes at JoVE.com

Assessment of Mitochondrial Functions and Cell Viability in Renal Cells Overexpressing Protein Kinase C Isozymes

호흡과 산화 인산화와와 세포 생존에 관련된 mitochondrial 기능에 단백질 키나제 C (PKC) isozymes의 활성화 효과가 설명되어 있습니다. 접근 방식은 선택적으로 mitochondrial 기능과 세포의 에너지 상태를 결정하는 주요 세포 배양 및 assays의 다양한 PKC isozymes을 overexpress하는 adenoviral 기술을 적응.

신장 세포 Overexpressing 단백질 키나제 C Isozymes에 Mitochondrial 기능 및 세포 생존 능력의 평가

The protein kinase C (PKC) family of isozymes is involved in numerous physiological and pathological processes. Our recent data demonstrate that PKC ...

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Biology

18.1K 조회수.  University of Arkansas for Medical Sciences. 호흡과 산화 인산화와와 세포 생존에 관련된 mitochondrial 기능에 단백질 키나제 C (PKC) isozymes의 활성화 효과가 설명되어 있습니다. 접근 방식은 선택적으로 mitochondrial 기능과 세포의 에너지 상태를 결정하는 주요 세포 배양 및 assays의 다양한 PKC isozymes을 overexpress하는 adenoviral 기술을 적응.

비디오: Assessment of Mitochondrial Functions and Cell Viability in Renal Cells Overexpressing Protein Kinase C Isozymes

Experimental Protocol for Detecting Mitochondrial Function in Hepatocytes Exposed to Organochlorine Pesticides

This paper presents detailed methods on detecting hepatic mitochondrial function for a better understanding the cause of metabolic disorders caused by environmental organochlorine pesticides (OCPs) in hepatocytes. HepG2 cells were exposed to &#946;-hexachlorocyclohexane (&#946;-HCH) for 24 h at an equivalent dose of internal exposure in general population. Ultrastructure in hepatocytes was examined by transmission electron microscopy (TEM) to show the damage of mitochondria. Mitochondrial function was further evaluated by mitochondrial fluorescence intensity, adenosine 5'-triphosphate (ATP) levels, oxygen consumption rate (OCR) and mitochondrial membrane potential (MMP) in HepG2 cells incubated with &#946;-HCH. The mitochondria fluorescence intensity after stained by mitochondrial green fluorescent probe was observed with a fluorescence microscopy. The luciferin-luciferase reaction was used to determine ATP levels. The MMP was detected by the cationic dye JC-1 and analyzed under flow cytometry. OCR was measured with an extracellular flux analyzer. In summary, these protocols were used in detecting mitochondrial function in hepatocytes with to investigate mitochondria damages.

Understanding the influence of environmental organochlorine pesticides (OCPs) on mitochondrial function in hepatocytes is important in exploring the mechanism of OCPs causing metabolic disorders. This paper presents detailed methods on detecting hepatic mitochondrial function.

유기체 살충제에 노출된 간세포에서 미토콘드리아 기능을 검출하기 위한 실험 프로토콜

This paper presents detailed methods on detecting hepatic mitochondrial function for a better understanding the cause of metabolic disorders caused by ...

연구

환경과학

DHODH 및 SDH 활성을 측정하기 위한 안정 동위원소 추적 및 LC-MS 분석

Oxygen-Independent Assays to Measure Mitochondrial Function in Mammals

The flow of electrons in the mitochondrial electron transport chain (ETC) supports multifaceted biosynthetic, bioenergetic, and signaling functions in mammalian cells. As oxygen (O2) is the most ubiquitous terminal electron acceptor for the mammalian ETC, the O2 consumption rate is frequently used as a proxy for mitochondrial function. However, emerging research demonstrates that this parameter is not always indicative of mitochondrial function, as fumarate can be employed as an alternative electron acceptor to sustain mitochondrial functions in hypoxia. This article compiles a series of protocols that allow researchers to measure mitochondrial function independently of the O2 consumption rate. These assays are particularly useful when studying mitochondrial function in hypoxic environments. Specifically, we describe methods to measure mitochondrial ATP production, de novo pyrimidine biosynthesis, NADH oxidation by complex I, and superoxide production. In combination with classical respirometry experiments, these orthogonal and economical assays will provide researchers with a more comprehensive assessment of mitochondrial function in their system of interest.

저자 스포트라이트: 포유류의 미토콘드리아 기능을 측정하기 위한 산소 독립적 분석  

Here, we present a compilation of assays to directly measure mitochondrial function in mammalian cells independently of their ability to consume molecular oxygen.

포유류의 미토콘드리아 기능을 측정하기 위한 산소 비의존적 분석

The flow of electrons in the mitochondrial electron transport chain (ETC) supports multifaceted biosynthetic, bioenergetic, and signaling functions in ...

생화학

Evaluation of Bioenergetic Function in Cerebral Vascular Endothelial Cells

The integrity of the blood-brain-barrier (BBB) is critical to prevent brain injury. Cerebral vascular endothelial (CVE) cells are one of the cell types that comprise the BBB; these cells have a very high-energy demand, which requires optimal mitochondrial function. In the case of disease or injury, the mitochondrial function in these cells can be altered, resulting in disease or&#160;the opening of the BBB. In this manuscript, we introduce a method to measure mitochondrial function in CVE cells by using whole, intact cells and a bioanalyzer. A mito-stress assay is used to challenge the cells that have been perturbed, either physically or chemically, and evaluate their bioenergetic function. Additionally, this method also provides a useful way to screen new therapeutics that have direct effects on mitochondrial function. We have optimized the cell density necessary to yield oxygen consumption rates that allow for the calculation of a variety of mitochondrial parameters, including ATP production, maximal respiration, and spare capacity. We also show the sensitivity of the assay by demonstrating that the introduction of the&#160;microRNA, miR-34a, leads to a pronounced and detectable decrease in mitochondrial activity. While the data shown in this paper is optimized for the bEnd.3 cell line, we have also optimized the protocol for primary CVE cells, further suggesting the utility in preclinical and clinical models.

Endothelial cell mitochondria are critical to maintain blood-brain-barrier integrity. We introduce a protocol to measure bioenergetic function in cerebral vascular endothelial cells.

대뇌 혈관 내피 세포의 생체 에너지 기능의 평가

The integrity of the blood-brain-barrier (BBB) is critical to prevent brain injury. Cerebral vascular endothelial (CVE) cells are one of the cell types ...

신경과학

Real-time Monitoring of Mitochondrial Respiration in Cytokine-differentiated Human Primary T Cells

During activation, the metabolism of T cells adapts to changes that impact their fate. An increase in mitochondrial oxidative phosphorylation is indispensable for T cell activation, and the survival of memory T cells is dependent on mitochondrial remodeling. Consequently, this affects the long-term clinical outcome of cancer immunotherapies. Changes in T cell quality are often studied by flow cytometry using well-known surface markers and not directly by their metabolic state. This is an optimized protocol for measuring real-time mitochondrial respiration of primary human T cells using an Extracellular Flux Analyzer and the cytokines IL-2 and IL-15, which differently affect T cell metabolism. It is shown that the metabolic state of T cells can clearly be distinguished by measuring the oxygen consumption when inhibiting key complexes in the metabolic pathway and that the accuracy of these measurements is highly dependent on optimal inhibitor concentration and inhibitor injection strategy. This standardized protocol will help implement mitochondrial respiration as a standard for T cell fitness in monitoring and studying cancer immunotherapies.

Metabolic adaptation is fundamental for T cells as it dictates differentiation, persistence, and cytotoxicity. Here, an optimized protocol for monitoring mitochondrial respiration in ex vivo cytokine-differentiated human primary T cells is presented.

사이토카인 분화된 인간 일차 T 세포에서 미토콘드리아 호흡의 실시간 모니터링

During activation, the metabolism of T cells adapts to changes that impact their fate. An increase in mitochondrial oxidative phosphorylation is ...

면역학 및 감염병학

Methods to Assess Subcellular Compartments of Muscle in C. elegans

Muscle is a dynamic tissue that responds to changes in nutrition, exercise, and disease state. The loss of muscle mass and function with disease and age are significant public health burdens. We currently understand little about the genetic regulation of muscle health with disease or age. The nematode C. elegans is an established model for understanding the genomic regulation of biological processes of interest. This worm&#8217;s body wall muscles display a large degree of homology with the muscles of higher metazoan species. Since C. elegans is a transparent organism, the localization of GFP to mitochondria and sarcomeres allows visualization of these structures in vivo. Similarly, feeding animals cationic dyes, which accumulate based on the existence of a mitochondrial membrane potential, allows the assessment of mitochondrial function in vivo. These methods, as well as assessment of muscle protein homeostasis, are combined with assessment of whole animal muscle function, in the form of movement assays, to allow correlation of sub-cellular defects with functional measures of muscle performance. Thus, C. elegans provides a powerful platform with which to assess the impact of mutations, gene knockdown, and/or chemical compounds upon muscle structure and function. Lastly, as GFP, cationic dyes, and movement assays are assessed non-invasively, prospective studies of muscle structure and function can be conducted across the whole life course and this at present cannot be easily investigated in vivo in any other organism.

Methods to Assess Subcellular Compartments of Muscle in C. elegans

Skeletal muscle is essential for locomotion and is the bodies&#8217; main protein store. Muscle health measurements within C. elegans are described. Prospective changes to muscle structure and function are assessed using localized GFP and cationic dyes.

방법은 근육의 세포 내 구획을 평가하기 위해<em&gt; C. 엘레</em

Muscle is a dynamic tissue that responds to changes in nutrition, exercise, and disease state. The loss of muscle mass and function with disease and age ...

생물학

Isolation and Functional Analysis of Mitochondria from Cultured Cells and Mouse Tissue

Comparison between two or more distinct groups, such as healthy vs. disease, is necessary to determine cellular status. Mitochondria are at the nexus of cell heath due to their role in both cell metabolism and energy production as well as control of apoptosis. Therefore, direct evaluation of isolated mitochondria and mitochondrial perturbation offers the ability to determine if organelle-specific (dys)function is occurring. The methods described in this protocol include isolation of intact, functional mitochondria from HEK cultured cells and mouse liver and spinal cord, but can be easily adapted for use with other cultured cells or animal tissues. Mitochondrial function assessed by TMRE and the use of common mitochondrial uncouplers and inhibitors in conjunction with a fluorescent plate reader allow this protocol not only to be versatile and accessible to most research laboratories, but also offers high throughput.

Comparison of mitochondrial membrane potential between samples yields valuable information about cellular status. Detailed steps for isolating mitochondria and assessing response to inhibitors and uncouplers using fluorescence are described. The method and utility of this protocol are illustrated by use of a cell culture and animal model of cellular stress.

분리 및 배양 세포 및 마우스 조직에서 미토콘드리아의 기능적 분석

Comparison between two or more distinct groups, such as healthy vs. disease, is necessary to determine cellular status. Mitochondria are at the nexus of ...

Experimental Approaches to Study Mitochondrial Localization and Function of a Nuclear Cell Cycle Kinase, Cdk1

Although mitochondria possess their own transcriptional machinery, merely 1% of mitochondrial proteins are synthesized inside the organelle. The nuclear-encoded proteins are transported into mitochondria guided by their mitochondria targeting sequences (MTS); however, a majority of mitochondrial localized proteins lack an identifiable MTS. Nevertheless, the fact that MTS can instruct proteins to go into the mitochondria provides a valuable tool for studying mitochondrial functions of normally nuclear and/or cytoplasmic proteins. We have recently identified the cell cycle kinase CyclinB1/Cdk1 complex in the mitochondria. To specifically study the mitochondrial functions of this complex, mitochondrial overexpression and knock-down of this complex without interfering with its nuclear or cytoplasmic functions were essential. By tagging CyclinB1/Cdk1 with MTS, we were able to achieve mitochondrial overexpression of this complex to study its mitochondrial targets as well as functions. Via tagging dominant-negative Cdk1 with MTS, inhibition of Cdk1 activity was accomplished particularly in the mitochondria. Potential mitochondrial targets of CyclinB1/Cdk1 complex were identified using a gel-based proteomics approach. Unlike traditional 2D gel analysis, we employed 2-dimensional difference gel electrophoresis (2D-DIGE) technology followed by phosphoprotein staining to fluorescently label differentially phosphorylated proteins in mitochondrial Cdk1 expressing cells. Identification of phosphoprotein spots that were altered in wild type versus dominant negative Cdk1 bearing mitochondria revealed the identity of mitochondrial targets of Cdk1. Finally, to determine the effect of CyclinB1/Cdk1 mitochondrial localization in cell cycle progression, a cell proliferation assay using a synthetic thymidine analogue EdU (5-ethynyl-2&#8242;-deoxyuridine) was used to monitor the cells as they go through the cell cycle and replicate their DNA. Altogether, we demonstrated a variety of approaches available to study mitochondrial localization and activity of a cell cycle kinase. These are advanced, yet easy to follow methods that will be beneficial to many cell biology researchers.

Here, we outline how to study mitochondrial localization of a (cell cycle) kinase, and how to determine its sub-mitochondrial location as well as potential mitochondrial substrates/targets. Forced expression of proteins into the mitochondria provides a useful tool for studying the functional consequences of mitochondrial localization of a protein of interest.

실험 접근법은 미토콘드리아 현지화 및 핵 세포주기 키나제,는 Cdk1의 기능을 연구하는

Although mitochondria possess their own transcriptional machinery, merely 1% of mitochondrial proteins are synthesized inside the organelle. The ...

Isolation, Characterization, And High Throughput Extracellular Flux Analysis of Mouse Primary Renal Tubular Epithelial Cells

Mitochondrial dysfunction in the renal tubular epithelial cells (TECs) can lead to renal fibrosis, a major cause of&#160;chronic kidney disease (CKD). Therefore, assessing mitochondrial function in primary TECs may provide valuable insight into the bioenergetic status of the cells, providing insight into the pathophysiology of CKD. While there are a number of complex protocols available for the isolation and purification of proximal tubules in different species, the field lacks a cost-effective method optimized for&#160;tubular cell isolation without the need for purification. Here, we provide an isolation protocol that allows for studies focusing on both primary mouse proximal and distal renal TECs. In addition to cost-effective reagents and minimal animal procedures required in this protocol, the isolated cells maintain high energy levels after isolation and can be sub-cultured up to four&#160;passages, allowing for continuous studies. Furthermore, using a high throughput extracellular flux analyzer, we assess the mitochondrial respiration directly in the isolated TECs in a 96-well plate for which we provide recommendations for the optimization of cell density and compound concentration. These observations suggest that this protocol can be used for renal tubular ex vivo studies with a consistent, well-standardized production of renal TECs. This protocol may have broader future applications to study mitochondrial dysfunction associated with renal disorders for drug discovery or drug characterization purposes.

This protocol provides a cost-effective approach to isolate and characterize mouse primary renal tubular cells that can subsequently be sub-cultured to assess renal biological functions ex vivo, including mitochondrial bioenergetics.

마우스 기본 신장 관 상피 세포의 고립, 특성, 및 높은 처리량 세포 외 유출 분석

Mitochondrial dysfunction in the renal tubular epithelial cells (TECs) can lead to renal fibrosis, a major cause of&#160;chronic kidney disease (CKD). ...

신장 세포 Overexpressing 단백질 키나제 C Isozymes에 Mitochondrial 기능 및 세포 생존 능력의 평가

요약

더 많은 비디오 탐색

이 비디오의 챕터

유기체 살충제에 노출된 간세포에서 미토콘드리아 기능을 검출하기 위한 실험 프로토콜

포유류의 미토콘드리아 기능을 측정하기 위한 산소 비의존적 분석

포유류의 미토콘드리아 기능을 측정하기 위한 산소 비의존적 분석

포유류의 미토콘드리아 기능을 측정하기 위한 산소 비의존적 분석

대뇌 혈관 내피 세포의 생체 에너지 기능의 평가

사이토카인 분화된 인간 일차 T 세포에서 미토콘드리아 호흡의 실시간 모니터링

방법은 근육의 세포 내 구획을 평가하기 위해

분리 및 배양 세포 및 마우스 조직에서 미토콘드리아의 기능적 분석

실험 접근법은 미토콘드리아 현지화 및 핵 세포주기 키나제,는 Cdk1의 기능을 연구하는

마우스 기본 신장 관 상피 세포의 고립, 특성, 및 높은 처리량 세포 외 유출 분석