この作品は、米国国立衛生研究所、国立糖尿病の研究所および消化器腎臓病、2R01DK59558（GNへの）からの助成金によってサポートされていました。研究資源助成UL1 RR029884のための健康のナショナルセンターの国立研究所によってサポートUAMSトランスレーショナル·リサーチ研究所がUAMSでフローサイトメトリーコアの部分的な資金を提供した。私たちは感謝博士Peipei平（カリフォルニア大学ロサンゼルス校、ロサンゼルス、カリフォルニア州）のcDNAのPKC-εと博士アレンSamarel（ロヨラ大学医療センターのドミナントネガティブ（非アクティブ）の変異体コーディング運ぶアデノウイルスのアリコートを提供するため; PKC-εの恒常的活性変異体をコードするアデノウイルスベクターのアリコートを提供するためのメイウッド、イリノイ州）。我々はまた、博士に感謝します。 PKC-ε恒常的活性をコードするcDNAを提供するためのピーター·パーカーとピーターサグデン（インペリアル·カレッジ·ロンドン、ロンドン、英国）。

<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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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>

特別な利害関係は宣言されません。

ここで紹介する手法は、腎近位尿細管細胞の初代培養におけるPKCの個々のアイソザイムの過剰発現を可能にします。このアプローチにはいくつかの長所があります：1。それは様々な侮辱（虚血、低酸素、酸化ストレス）、薬、腎臓内nephrotoxicantsのための第一の目標である細胞（腎近位尿細管細胞）の均一な集団での調節機構を調査することができます。 2。改良された条件で初代培養で増殖?...

<table border="1"><tbody><tr><td> 試薬の名称 </td><td> 会社 </td><td> カタログ番号 </td></tr><tr><td>層流フード</td><td>サーモエレクトロン株式会社</td><td> FORMA 1104 </td></tr><tr><td> 2ミリリットルと15ミリリットルのダウンス組織グラインダー</td><td> WHEATON </td><td> 989から24607、357544 </td></tr><tr><td> 85〜235ミクロンのナイロンメッシュ</td><td>小型部品</td><td> CMN - 0085 - 10YD CMN - 0250 - 10YD </td></tr><tr><td> 50ミリリットル滅菌遠心管</td><td> BIOLOGIX </td><td> BCT-P 50BS </td></tr><tr><td> 1.5 mlマイクロチューブ</td><td>ザルスタット</td><td> 72.690.001 </td></tr><tr><td> 35 x 10 mmの無菌培養皿</td><td>コー​​ニング</td><td> 430165 </td></tr><tr><td>ジョアン遠心機</td><td>ジョアン</td><td>ジョアンCR3 11175704ローター：ジョアンT40 </td></tr><tr&gt; <td>調整可能なマイクロ遠心</td><td> SIGMA </td><td>モデル1から15 </td></tr><tr><td>生物学的酸素モニタ</td><td> YSI Incorporatedの</td><td> YSIモデル5300A </td></tr><tr><td>シングルチャンバマイクロ酸素システム</td><td> YSI Incorporatedの</td><td> 5356S </td></tr><tr><td>酸素プローブ</td><td> YSI Incorporatedの</td><td> 5331A </td></tr><tr><td>循環バス</td><td> YSI Incorporatedの</td><td> 5310 </td></tr><tr><td> KClおよび標準メンブレンキット</td><td> YSI Incorporatedの</td><td> 5775 </td></tr><tr><td>マグネチックスターラー</td><td> YSI Incorporatedの</td><td> 5222 </td></tr><tr><td>フラットベッドレコーダー</td><td>キップ＆Zonen </td><td> BD 11E </td></tr><tr><td> 48ウェルと96ウェル透明板</td><td>コスター</td><td> 3548、3679 </td></tr><tr><td>サーモR </td><td> Eppendorf </td><td> 5355 21919 </td></tr><tr><td>オービタルシェーカーMAXQ 2000 </td><td>サーモサイエンティフィック</td><td> SHKA 2000 </td></tr><tr><td>スペクトルFLUORプラス（吸光/蛍光/発光リーダー） </td><td>テカン</td><td> F129005 </td></tr><tr><td>ウォータージャケット米国オートフロー自動CO 2インキュベーター</td><td> NUAIRE </td><td> NU 4850 </td></tr><tr><td>フローサイトメトリー用の12x75 mmのポリスチレン培養試験管</td><td>フィッシャーブランド</td><td> 14-961-20 </td></tr><tr><td> Axioskop 水浸対物レンズ63X / 0,90 W </td><td>カールツァイス</td><td> 114846 ACHROPLAN 44 00 67 </td></tr><tr><td> DMEM / F12キー</td><td> Cellgro </td><td> 99から830 - PB </td></tr><tr><td> DMEM / F12のハム</td><td>シグマ</td><td> D 2906 - 1L </td></tr><tr><td>メシル酸デフェロキサミン</td><td> HospiRA </td><td> D110 </td></tr><tr><td>コラゲナーゼタイプI </td><td>ワージントン</td><td> 4196 </td></tr><tr><td>トリプシン阻害剤</td><td>シグマ</td><td> T 6522 - 500mgの</td></tr><tr><td> 5,5 &#39;、6,6&#39; - テトラクロロ-1,1 &#39;、3,3&#39; - tetraethylbenzimidazolylcarbocyanineヨウ（JC-1） </td><td>インビトロジェン</td><td> T3168 </td></tr><tr><td> Mitotracker赤</td><td>インビトロジェン</td><td> M22425 </td></tr><tr><td> ATPの生物発光アッセイキットHS II </td><td>ロッシュ</td><td> 11 699 709 001 </td></tr><tr><td>アネキシンV - FITCソリューション</td><td>バイオビジョン</td><td> 1001 - 200 </td></tr><tr><td>フローサイトメーター</td><td> BD Biosciences社</td><td> BD FACSキャリバー</td></tr></tbody></table>

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

アイソザイムのプロテインキナーゼC（PKC）ファミリーは多数の生理学的および病理学的プロセスに関与している。我々の最近のデータは、PKCは、ミトコンドリアの機能と細胞のエネルギー状態を調節していることを実証している。多数の報告は、PKC-αおよびPKC-εの活性化は虚血性心とを仲介する心保護におけるミトコンドリア機能を改善することを実証した。対照的に、我々は、PKC-α、PKC-εは腎細胞でnephrotoxicant誘発性ミトコンドリア機能障害や細胞死に関与していることを明らかにした。したがって、本研究の目的は、PKCアイソザイムを選択的に活性化または酸化的リン酸化と細胞生存の制御に果たす役割を決定するために阻害されるおそれのある積極的なミトコンドリアの機能を維持する腎細胞のin vitroモデルを開発することであった。腎近位尿細管細胞（RPTC）の初代培養は水戸のミトコンドリア呼吸と活動の結果改善された条件で培養したin vivoでの RPTCと同様chondrial酵素。伝統的なトランスフェクション技術（リポフェクタミン、エレクトロポレーション）は初代培養において非効率的であり、ミトコンドリアの機能に悪影響を及ぼすので、PKC-ε変異体cDNAをアデノウイルスベクターを介しRPTCに届けられました。 90％を超える培養R​​PTCのトランスフェクションでは、このアプローチは結果。 1：ここでは、におけるPKC-εの役割を評価するための方法を提示します。 ATP合成に関連付けられているミトコンドリアの形態と機能の調節、および2。初代培養におけるRPTCの生存。 PKC-εが過剰発現している恒常的に活性化、PKC-ε突然変異体によって活性化される。 PKC-εは、過剰発現することにより、PKC-εの不活性変異体を阻害される。ミトコンドリア機能は、呼吸、呼吸鎖の完全性、呼吸複合体およびF 0 F 1-ATPアーゼ、ATPの生産速度、およびATP含量の活動を調べることで評価される。呼吸がある状態3（過剰基質およびADPの存在下で最大呼吸）と非結合呼吸としてジギトニン透過性RPTCにssessed。呼吸鎖の完全性は、単離されたミトコンドリアの呼吸鎖のすべての4つの複合体の活性を測定することにより評価される。酸化的リン酸化の容量は、ミトコンドリア膜電位、ATPの生産速度、およびF 0 F 1-ATPaseの活性を測定することによって評価されます。 RPTCのエネルギー状態は、細胞内ATP含量を測定することにより評価されています。生きた細胞内のミトコンドリアの形態はMitoTracker赤580、具体的にミトコンドリアに蓄積蛍光染料、およびライブ単層蛍光顕微鏡で調べたを用いて可視化されています。 RPTC生存率は、アネキシンV /アポトーシスと腫瘍症を決定するために、フローサイトメトリーに続いてヨウ化プロピジウム染色を用いて評価されています。 これらの方法は、個々のPKCアイソザイムの選択的活性化/阻害を可能にin vitroで再生することができる生理学的、病理学的条件の様々な細胞機能における役割を評価した。

Figure 1に示すとおりのアデノウイルスデリバリーRPTCとミトコンドリアにおけるPKC-εの大幅に増加したタンパク質レベルにおけるPKC-ε結果の恒常的活性（caPKC-ε）と非アクティブ（dnPKC - ε）変異体をコードするcDNA。細胞が不活性であったcDNAコードdnPKCを過剰発現- εPKC-ε（リン酸化されていない）（ 図1）に感染したのに対し、caPKC-εベクターを有するcDNAを感染させた?...

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

呼吸と酸化的リン酸化により、細胞生存率に関連するミトコンドリア機能に対するプロテインキナーゼC（PKC）アイソザイムの活性化の効果を説明する。アプローチは、選択的に初代細胞培養や細胞のミトコンドリアの機能とエネルギー状態を決定するために種々のアッセイにおけるPKCアイソザイムを過剰発現するアデノウイルスの手法を適応します。

腎細胞内のミトコンドリアの機能と細胞生存率の評価を過剰発現するプロテインキナーゼCアイソザイム

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

assessment-mitochondrial-functions-cell-viability-renal-cells

Research

JoVE Journal

Biology

18.1K ビュー.  University of Arkansas for Medical Sciences. 呼吸と酸化的リン酸化により、細胞生存率に関連するミトコンドリア機能に対するプロテインキナーゼC（PKC）アイソザイムの活性化の効果を説明する。アプローチは、選択的に初代細胞培養や細胞のミトコンドリアの機能とエネルギー状態を決定するために種々のアッセイにおけるPKCアイソザイムを過剰発現するアデノウイルスの手法を適応します。

ビデオ: 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.

シトクロムの細胞質マイクロインジェクションによるアポトーシスの活性化 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 ...

生物学

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.

タリイメージベースのメ​​ーターを用いたGFPの発現と生存率の評価

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.

生細胞中のタンパク質凝集の4Dイメージング

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.

培養中の細胞のための生存率アッセイ

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.

ミトコンドリア局在と原子力細胞周期キナーゼ、Cdk1のの機能を研究するための実験的アプローチ

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.

蛍光顕微鏡による生細胞内のミトコンドリアのカルシウムとミトコンドリア膜電位の同時測定

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.

動的細胞突起におけるソフトウェア支援トラッキングのためのグラフィカルユーザインタフェース

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.

初代培養ラット副腎皮質細胞とステロイド ホルモン産生機能の試金

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.

フェムトセカンドレーザーによる光刺激は、標的細胞における細胞外シグナル調節キナーゼ(ERK)シグナル伝達またはミトコンドリアイベントを活性化する

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.

流出ポンプの阻害による造血幹細胞および前駆細胞におけるミトコンドリア膜電位の流れ細胞測定評価の精度向上

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