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. 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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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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

Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c

Apoptosis, or programmed cell death, is a conserved and highly regulated pathway by which cells die1. Apoptosis can be triggered when cells encounter a wide range of cytotoxic stresses. These insults initiate signaling cascades that ultimately cause the release of cytochrome c from the mitochondrial intermembrane space to the cytoplasm2. The release of cytochrome c from mitochondria is a key event that triggers the rapid activation of caspases, the key cellular proteases which ultimately execute cell death3-4.
The pathway of apoptosis is regulated at points upstream and downstream of cytochrome c release from mitochondria5. In order to study the post-mitochondrial regulation of caspase activation, many investigators have turned to direct cytoplasmic microinjection of holocytochrome c (heme-attached) protein into cells6-9. Cytochrome c is normally localized to the mitochondria where attachment of a heme group is necessary to enable it to activate apoptosis10-11. Therefore, to directly activate caspases, it is necessary to inject the holocytochrome c protein instead of its cDNA, because while the expression of cytochrome c from cDNA constructs will result in mitochondrial targeting and heme attachment, it will be sequestered from cytosolic caspases. Thus, the direct cytosolic microinjection of purified heme-attached cytochrome c protein is a useful tool to mimic mitochondrial cytochrome c release and apoptosis without the use of toxic insults which cause cellular and mitochondrial damage.
In this article, we describe a method for the microinjection of cytochrome c protein into cells, using mouse embryonic fibroblasts (MEFs) and primary sympathetic neurons as examples. While this protocol focuses on the injection of cytochrome c for investigations of apoptosis, the techniques shown here can also be easily adapted for microinjection of other proteins of interest.

Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c

In this protocol, we describe the direct cytoplasmic microinjection of cytochrome c protein into fibroblasts and primary sympathetic neurons. This technique allows for the introduction of cytochrome c protein into the cytoplasm of cells and mimics the release of cytochrome c from mitochondria, which occurs during apoptosis.

시토크롬의 세포질 Microinjection 의한 Apoptosis의 활성화 C</em

Apoptosis, or programmed cell death, is a conserved and highly regulated pathway by which cells die1.  Apoptosis can be triggered when cells encounter a ...

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생물학

Assessment of GFP Expression and Viability Using the Tali Image-Based Cytometer

Single-cell and population information are commonly obtained either by flow cytometry or fluorescence microscopy. However, these two methods provide different information. Flow cytometry gives quantitative multi-parametric information about physical characteristics and staining or expression, but doesn't allow for visualization. Stand-alone fluorescence microscopy provides visual data, but doesn't allow for straightforward quantitative measurements1.
Image-based cytometry bridges the gap between these two methods, enabling the quick visualization and simultaneous quantitative analysis of thousands of cells in heterogeneous populations2. Here, we present a method for performing cell viability and green fluorescent protein (GFP) expression assays using the Tali Image-Based Cytometer3. The Tali instrument is a 3-channel (bright field, green fluorescence, red fluorescence) benchtop assay platform that offers several advantages over flow cytometry and fluorescence microscopy. The Tali cytometer is less expensive, takes up less bench space, requires less maintenance, and the work flow has been simplified so that the operation and analysis is much simpler and quicker. The Tali cytometer is capable of performing a range of suspension cell-based assays, including GFP and red fluorescent protein (RFP) expression, apoptosis4-6 and cell viability analysis with propidium iodide (PI)7-11.
Here, we demonstrate the use of the Tali instrument in performing a cell viability assay in cells expressing GFP. GFP-transduced cells are stained using the Tali Viability Kit - Dead Cell Red. The cells are then pipetted into a Tali Cellular Analysis Slide and loaded into the cytometer. Bright field, red fluorescence and green fluorescence images are captured and analyzed using assay specific algorithms. Histograms are then generated to display cell size, PI fluorescence intensity, and GFP fluorescence intensity. These parameters can then be thresholded to home in on a specific cell population.
A side-by side comparison of the Tali Image-Based Cytometer and traditional flow cytometry demonstrates that the two methods provide comparable data regarding cell viability and protein expression. However, the Tali instrument provides additional visual information about the cell population that cannot be obtained using a flow cytometer.

This protocol describes how to perform cell viability and fluorescence expression assays using the Tali Image-Based Cytometer.

Tali 이미지 기반 Cytometer를 사용하여 GFP의 표현과 생존 능력의 평가

Single-cell and population information are commonly obtained either by flow cytometry or fluorescence microscopy. However, these two methods provide ...

4D Imaging of Protein Aggregation in Live Cells

One of the key tasks of any living cell is maintaining the proper folding of newly synthesized proteins in the face of ever-changing environmental conditions and an intracellular environment that is tightly packed, sticky, and hazardous to protein stability1. The ability to dynamically balance protein production, folding and degradation demands highly-specialized quality control machinery, whose absolute necessity is observed best when it malfunctions. Diseases such as ALS, Alzheimer's, Parkinson's, and certain forms of Cystic Fibrosis have a direct link to protein folding quality control components2, and therefore future therapeutic development requires a basic understanding of underlying processes. Our experimental challenge is to understand how cells integrate damage signals and mount responses that are tailored to diverse circumstances.
The primary reason why protein misfolding represents an existential threat to the cell is the propensity of incorrectly folded proteins to aggregate, thus causing a global perturbation of the crowded and delicate intracellular folding environment1. The folding health, or "proteostasis," of the cellular proteome is maintained, even under the duress of aging, stress and oxidative damage, by the coordinated action of different mechanistic units in an elaborate quality control system3,4. A specialized machinery of molecular chaperones can bind non-native polypeptides and promote their folding into the native state1, target them for degradation by the ubiquitin-proteasome system5, or direct them to protective aggregation inclusions6-9.
In eukaryotes, the cytosolic aggregation quality control load is partitioned between two compartments8-10: the juxtanuclear quality control compartment (JUNQ) and the insoluble protein deposit (IPOD) (Figure 1 - model). Proteins that are ubiquitinated by the protein folding quality control machinery are delivered to the JUNQ, where they are processed for degradation by the proteasome. Misfolded proteins that are not ubiquitinated are diverted to the IPOD, where they are actively aggregated in a protective compartment.
Up until this point, the methodological paradigm of live-cell fluorescence microscopy has largely been to label proteins and track their locations in the cell at specific time-points and usually in two dimensions. As new technologies have begun to grant experimenters unprecedented access to the submicron scale in living cells, the dynamic architecture of the cytosol has come into view as a challenging new frontier for experimental characterization. We present a method for rapidly monitoring the 3D spatial distributions of multiple fluorescently labeled proteins in the yeast cytosol over time. 3D timelapse (4D imaging) is not merely a technical challenge; rather, it also facilitates a dramatic shift in the conceptual framework used to analyze cellular structure.
We utilize a cytosolic folding sensor protein in live yeast to visualize distinct fates for misfolded proteins in cellular aggregation quality control, using rapid 4D fluorescent imaging. The temperature sensitive mutant of the Ubc9 protein10-12 (Ubc9ts) is extremely effective both as a sensor of cellular proteostasis, and a physiological model for tracking aggregation quality control. As with most ts proteins, Ubc9ts is fully folded and functional at permissive temperatures due to active cellular chaperones. Above 30 &deg;C, or when the cell faces misfolding stress, Ubc9ts misfolds and follows the fate of a native globular protein that has been misfolded due to mutation, heat denaturation, or oxidative damage. By fusing it to GFP or other fluorophores, it can be tracked in 3D as it forms Stress Foci, or is directed to JUNQ or IPOD.

Cellular viability depends on timely and efficient management of protein misfolding. Here we describe a method for visualizing the different potential fates of a misfolded protein: refolding, degradation, or sequestration in inclusions. We demonstrate the use of a folding sensor, Ubc9ts, for monitoring proteostasis and aggregation quality control in live cells using 4D microscopy.

라이브 셀의 단백질 응집체의 4D 영상

One of the key tasks of any living cell is maintaining the proper folding of newly synthesized proteins in the face of ever-changing environmental ...

Viability Assays for Cells in Culture

Manual cell counts on a microscope are a sensitive means of assessing cellular viability but are time-consuming and therefore expensive. Computerized viability assays are expensive in terms of equipment but can be faster and more objective than manual cell counts. The present report describes the use of three such viability assays. Two of these assays are infrared and one is luminescent. Both infrared assays rely on a 16&#160;bit Odyssey Imager. One infrared assay uses the DRAQ5 stain for nuclei combined with the Sapphire stain for cytosol and is visualized in the 700 nm channel. The other infrared assay, an In-Cell Western, uses antibodies against cytoskeletal proteins (&#945;-tubulin or microtubule associated protein 2) and labels them in the 800 nm channel. The third viability assay is a commonly used luminescent assay for ATP, but we use a quarter of the recommended volume to save on cost. These measurements are all linear and correlate with the number of cells plated, but vary in sensitivity. All three assays circumvent time-consuming microscopy and sample the entire well, thereby reducing sampling error. Finally, all of the assays can easily be completed within one day of the end of the experiment, allowing greater numbers of experiments to be performed within short timeframes. However, they all rely on the assumption that cell numbers remain in proportion to signal strength after treatments, an assumption that is sometimes not met, especially for cellular ATP. Furthermore, if cells increase or decrease in size after treatment, this might affect signal strength without affecting cell number. We conclude that all viability assays, including manual counts, suffer from a number of caveats, but that computerized viability assays are well worth the initial investment. Using all three assays together yields a comprehensive view of cellular structure and function.

Therapeutic compounds are often first examined in vitro with viability assays. Blind cell counts by a human observer can be highly sensitive to small changes in cell number but do not assess function. Computerized viability assays, as described here, can assess both structure and function in an objective manner.

문화의 세포에 대한 생존 분석 실험

Manual cell counts on a microscope are a sensitive means of assessing cellular viability but are time-consuming and therefore expensive. Computerized ...

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 ...

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+ ...

A Graphical User Interface for Software-assisted Tracking of Protein Concentration in Dynamic Cellular Protrusions

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex signaling mechanisms underlying filopodial initiation, elongation and subsequent stabilization or retraction, it is crucial to determine the spatio-temporal protein activity in these dynamic structures. To analyze protein function in filopodia, we recently developed a semi-automated tracking algorithm that adapts to filopodial shape-changes, thus allowing parallel analysis of protrusion dynamics and relative protein concentration along the whole filopodial length. Here, we present a detailed step-by-step protocol for optimized cell handling, image acquisition and software analysis. We further provide instructions for the use of optional features during image analysis and data representation, as well as troubleshooting guidelines for all critical steps along the way. Finally, we also include a comparison of the described image analysis software with other programs available for filopodia quantification. Together, the presented protocol provides a framework for accurate analysis of protein dynamics in filopodial protrusions using image analysis software.

We present a software solution for semi-automated tracking of relative protein concentration along the length of dynamic cellular protrusions.

동적 세포 돌출부에서 소프트웨어를 이용한 단백질 농도 추적을위한 그래픽 사용자 인터페이스

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex ...

Primary Culture of Rat Adrenocortical Cells and Assays of Steroidogenic Functions

The hormone which the adrenal cortex secretes is vital for animals against stress and diseases. The method described here is the procedure of primary cultured rat adrenal cells and related functional assays (immunofluorescence staining of lipid droplet surface protein, as well as corticosterone analysis). Unlike an in vivo model, the variation of interexperiments in adrenal monolayer cultures is less and the experimental condition is easy to control. Besides, the source of rats is also more stable than other animals, like bovine ones. There are also several human adrenal cell lines (NCI-H295, NCI-H295R, SW13, etc.) that can be used in adrenal studies. However, the steroid production of these lines will still be influenced by numerous factors, which include serum lot number, passage number, mutant/loss of distinct genes, etc. Except for lacking 17&#945;-hydroxylase, the primary culture of rat adrenocortical cells is a better and more convenient technique for studying adrenal physiology. In summary, primary rat adrenal cultures could be a good in vitro platform for researchers to investigate the mechanisms of the reagent of interest in the adrenal gland system.

The hormone secreted by the adrenal cortex is vital for animals against stress and diseases. Here, we present a protocol to culture the primary rat adrenal cells. It could be a good in vitro platform for investigating the mechanisms of the reagent of interest in adrenal steroidogenesis and lipid biosynthesis.

쥐 Adrenocortical 셀 및 Steroidogenic 기능의 분석 실험의 기본 문화

The hormone which the adrenal cortex secretes is vital for animals against stress and diseases. The method described here is the procedure of primary ...

Photostimulation by Femtosecond Laser Activates Extracellular-signal-regulated Kinase (ERK) Signaling or Mitochondrial Events in Target Cells

Direct control of cellular defined molecular events is important to life science. Recently, studies have demonstrated that femtosecond laser stimulation can simultaneously activate multiple cellular molecular signaling pathways. In this protocol, we show that through coupling femtosecond laser into a confocal microscope, cells can be stimulated precisely by the tightly-focused laser. Some molecular processes that can be simultaneously observed are subsequently activated. We present detailed protocols of the photostimulation to activate extracellular signal regulated kinase (ERK) signaling pathway in Hela cells. Mitochondrial flashes of reactive oxygen species (ROS) and other mitochondrial events can be also stimulated if focusing the femtosecond laser pulse on a certain mitochondrial tubular structure. This protocol includes pretreating cells before photostimulation, delivering the photostimulation by a femtosecond laser flash onto the target, and observing/identifying molecular changes afterwards. This protocol represents an all-optical tool for related biological researches.

Photostimulation by Femtosecond Laser Activates Extracellular-signal-regulated Kinase ERK Signaling or Mitochondrial Events in Target Cells

Tightly-focused femtosecond laser can deliver precise stimulation to cells by being coupled into a confocal microscopy enabling the real-time observation and photostimulation. The photostimulation can activate cell molecular events including ERK signaling pathway and mitochondrial flashes of reactive oxygen species.

펨토초 레이저에 의한 광자극은 대상 세포에서 세포외 신호 조절 키나아제(ERK) 신호 또는 미토콘드리아 이벤트를 활성화합니다.

Direct control of cellular defined molecular events is important to life science. Recently, studies have demonstrated that femtosecond laser stimulation ...

Improving the Accuracy of Flow Cytometric Assessment of Mitochondrial Membrane Potential in Hematopoietic Stem and Progenitor Cells Through the Inhibition of Efflux Pumps

As cellular metabolism is a key regulator of hematopoietic stem cell (HSC) self-renewal, the various roles played by the mitochondria in hematopoietic homeostasis have been extensively studied by HSC researchers. Mitochondrial activity levels are reflected in their membrane potentials (&#916;&#936;m), which can be measured by cell-permeant cationic dyes such as TMRM (tetramethylrhodamine, methyl ester). The ability of efflux pumps to extrude these dyes from cells can limit their usefulness, however. The resulting measurement bias is particularly critical when assessing HSCs, as xenobiotic transporters exhibit higher levels of expression and activity in HSCs than in differentiated cells. Here, we describe a protocol utilizing Verapamil, an efflux pump inhibitor, to accurately measure &#916;&#936;m across multiple bone marrow populations. The resulting inhibition of pump activity is shown to increase TMRM intensity in hematopoietic stem and progenitor cells (HSPCs), while leaving it relatively unchanged in mature fractions. This highlights the close attention to dye-efflux activity that is required when &#916;&#936;m-dependent dyes are used, and as written and visualized, this protocol can be used to accurately compare either different populations within the bone marrow, or the same population across different experimental models.

Xenobiotic efflux pumps are highly active in hematopoietic stem and progenitor cells (HSPCs) and cause extrusion of TMRM, a mitochondrial membrane potential fluorescent dye. Here, we present a protocol to accurately measure mitochondrial membrane potential in HSPCs by TMRM in the presence of Verapamil, an efflux pump inhibitor.

에플로프 펌프 억제를 통해 조혈 줄기 및 전조 세포에서 미토콘드리아 막 전위성 유동 세포 측정 평가의 정확도 향상

As cellular metabolism is a key regulator of hematopoietic stem cell (HSC) self-renewal, the various roles played by the mitochondria in hematopoietic ...