The authors thank members of the Shapiro lab and Erin Schrader for comments on the manuscript. The authors acknowledge financial support from: NIH postdoctoral fellowship F32 GM100732 to JMS and NIH grants R01 GM51426 and R01 GM32506 to LS.

1.大規模同期性 - ウエスタンブロット、マイクロアレイ/ RNA-SEQ、およびその他素材集中アッセイに最適<ol><li>冷凍庫ストック又はプレートから、PYE培地で28℃で振とうすることにより歪NA1000の5ミリリットルO / N培養を成長。 </li><li> M2Gの25ミリリットル（表1-2）に、ステップ1からの細胞の0.5ミリリットルを接種し、培養が0.5と0.6の間のOD 600に達するまで28℃で振る。 </li><li> M2Gの1リットル中に細胞を接種し、28℃で振る。 </li><li> OD 600が 0.5〜0.6に達すると、液体取り付けられた位相差顕微鏡を用いて細胞swarmerの存在を確認する。位相差顕微鏡によってスライドガラス上の細胞の1μlを、カバーガラスで覆い、スポット、および画像。人口が急速に泳いで細胞を視覚化することによってswarmer細胞の存在を確認してください。 </li><li> JA-10ローターで4℃で7 kxgで15分間細胞をスピン。 </li><li>上清を捨て、冷たいM2（表1-2）の180ミリリットルを加え、穏やかにresは血清学的ピペットを使用してすべてのセルをuspend。緩くペレット化した細胞を捨てる。彼らはpredivisionalとストーカー細胞である。 </li><li>冷たいコロイダルシリカ溶液の60ミリリットルを加え、よく細胞懸濁液を混ぜる（よく細胞に添加する前にコロイダルシリカ懸濁液を混在してください）​​。 </li><li> 8 30ミリリットルチューブに細胞懸濁液を注ぎ、JA-20ローターで、4℃で6.4 kxgで30分間スピン。 注：Oneは、2つの異なるバンドが表示されるはずです。ストーカー/ predivisional細胞は、トップバンド（ 図1B）にいる間swarmer帯は下のバンドである。 </li><li>慎重にトップバンドをオフに吸引し、swarmerバンド（下のバンド）上記〜1 cmの液体を除去する。 </li><li>パスツールピペットを用いて（クリティカル）、慎重にswarmerバンドを削除して、清潔なチューブに配置します。 JA-20ローターで、4℃で6.4 kxgで10分間コロイダルシリカ、トップ冷たいM2とチューブオフとスピンを洗い流す。 </li><li>注意深く上清を破棄し、再懸濁JA-20ローターで冷たいM2の20ミリリットルと、4℃で6.4 kxgで10分間スピンのセル。 </li><li>コールドM2 30mlの中に全てのペレットを再懸濁し、分光光度計を用いてOD 600を測定し、冷M2培地を用いてブランク。 swarmer細胞を確認するために位相イメージングのための1μLを保存します。細胞90〜95％がswarmersでなければなりません。 </li><li> JA-20ローターで、4℃で6.4 kxgで5分間、細胞をスピンダウン。 28℃M2G培地中の細胞を再懸濁A 600になるように、〜0.3〜0.4と28℃で振とうを開始。 注：典型的な収量は30と同期していない細胞の1Lからswarmer細胞培養の60mLの間にある。 </li><li> 8,9（野生型の文化が分裂するのに約135〜140分かかります）の時点を取って開始します。各時点で、OD 600を測定する。分割後のOD 600が約2倍初期OD 600であることを確認してください。 </li><li>ウェスタンブロットまたは遺伝子発現アッセイのために、立方1mLのアリコートを除去所望の時点でlture、30秒間、卓上遠心分離機において最大速度でスピンダウンし、急速に培地をデカントまたは吸引し、フラッシュは、液体窒素中で細胞ペレットを凍結する。下流の分析まで-80℃で細胞を保管してください。 </li></ol> 2.小​​規模同期性 - 顕微鏡用に最適<ol><li>冷凍庫ストック又はプレートから、M2Gに28℃で振とう5ミリリットルO / N培養を成長。 </li><li> M2G（表1-2）15mlに希釈し、中期対数（OD 600 = 0.5〜0.6）になるまで成長する。 </li><li> JA-20ローター中で4℃で10分間6.4 kxgでスピンし、1ミリリットル中に再懸濁冷たいM2（表1-2）と2 mlのマイクロ遠心チューブに移す。 </li><li>上清を吸引し、細胞をペレットにマイクロ遠心チューブ中で3分間15 kxgでのスピンは、冷たいM2の900μlの氷の上でペレット、再懸濁を置く。 </li><li>マイクで4℃で15 kxgで20分間、コロイダルシリカとスピンコーティングされた冷たいPVP900μlのを追加します。rocentrifugeチューブ。 </li><li> （クリティカル）を吸引したり、トップを切っピペット/ predivisional細胞バンドをストーカーして、新しいマイクロ遠心チューブにバンドswarmer底に集める。 </li><li> 3分間15 kxgで遠心分離しながら、冷たいM2の1ミリリットル中swarmer細胞を2回洗浄します。 </li><li>最後のスピンの前に、予備冷却した1mlのガラス試験管に細胞を移動し、M2のブランクと比較した細胞のOD 600を測定する。 </li><li> 0.4及び28℃/ロール細胞を振る - 0.3の間ODで28℃、M2Gに最終的な細胞ペレットを再懸濁。 注：典型的な収量はswarmer細胞培養の2および4 mlの間である。 </li><li>顕微鏡実験のために、所望の時間にポイントがイメージングのためM2Gアガロースパッド上に細胞を1μLを置く。 </li></ol>

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The authors have nothing to disclose.

The bacterial cell cycle is a fundamental process in life and is important for the study of growth and as a target for next generation antibiotics. Here, we detailed the rapid synchronization procedures for C. crescentus NA1000, a model organism for the study of the bacterial cell cycle and asymmetric cell division. This method is amendable to western blot, gene expression profiling, and fluorescence microscopy assays to investigate the spatiotemporal regulation of the bacterial cell cycle. 
<p class='jove_...

細菌の細胞周期は、ゲノムの複製と娘細胞の分裂の両方を制御する。抗生物質耐性は、公衆の健康に成長している脅威であるとして重要なのは、細菌の細胞周期は、抗生物質開発のための未開発の目標を提示します。 細菌カウロバクターcrescentusでは、各細胞周期の異なる運命（ 図1A）1,2の2つの娘細胞を生じる、非対称分裂に導く。 One娘細胞は鞭毛を継承し、他の娘は茎を継承し、固着されている間運動性である。集積回路の遺伝子は、転写調節、リン酸化シグナル伝達、およびタンパク質分解規制3ことによって、細胞周期の進行および細胞の運命を制御する。また、染色体複製と同時分離は、第4染色体の正確に一つのコピーを含む娘細胞が得られる。重要なことに、これらの2つの細胞型は急速COLLOによって分離することができる高収率（ 図1B）を有する集団の残りからswarmer細胞の単離を可能にする同期可能NA1000株5-7 idalシリカ粒子の密度遠心分離。細胞s​​warmer分離された後、非対称細胞分裂を通じて同期的に進む。ここでは、詳細プロトコルは、 カウロバクター株 NA1000を同期させるために使用される。私たちは、プロトコルおよび大規模および小規模の両方の同期のための一般的なトラブルシューティングのヒントを提供する。この実験手順は、 カウロバクター細胞周期と細胞の運命の時空間制御を調べるための強力なツールを提供します。

<table border="0" cellpadding="0" cellspacing="0" width="573">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Name of Material/ Equipment</td>
			<td style="width:71px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">PVP Coated Colloidal Silica (Percoll)</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">P4937</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Colloidal Silica (Ludox AS-40)</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">420840</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">JA10 Rotor</td>
			<td style="width:71px;">Beckman-Coulter</td>
			<td style="width:119px;">369687</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">JA20 Rotor</td>
			<td style="width:71px;">Beckman-Coulter</td>
			<td style="width:119px;">334831</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Ferrous Sulfate Chelate Solution</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td>F0518</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">30 mL Centrifuge Tubes</td>
			<td style="width:71px;">Corning</td>
			<td style="width:119px;">8445</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:216px;">Na2HPO4</td>
			<td style="width:71px;">EMD</td>
			<td style="width:119px;">SX0720-1</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:216px;">KH2PO4</td>
			<td style="width:71px;">VWR</td>
			<td style="width:119px;">BDH9268-500G</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:216px;">NH4Cl</td>
			<td style="width:71px;">Amresco</td>
			<td style="width:119px;">0621-500g</td>
			<td></td>
		</tr>
	</tbody>
</table>

synchronization of caulobacter crescentus for investigation of the bacterial cell cycle

The cell cycle is important for growth, genome replication, and development in all cells. In bacteria, studies of the cell cycle have focused largely on unsynchronized cells making it difficult to order the temporal events required for cell cycle progression, genome replication, and division. Caulobacter crescentus provides an excellent model system for the bacterial cell cycle whereby cells can be rapidly synchronized in a G0 state by density centrifugation. Cell cycle synchronization experiments have been used to establish the molecular events governing chromosome replication and segregation, to map a genetic regulatory network controlling cell cycle progression, and to identify the establishment of polar signaling complexes required for asymmetric cell division. Here we provide a detailed protocol for the rapid synchronization of Caulobacter NA1000 cells. Synchronization can be performed in a large-scale format for gene expression profiling and western blot assays, as well as a small-scale format for microscopy or FACS assays. The rapid synchronizability and high cell yields of Caulobacter make this organism a powerful model system for studies of the bacterial cell cycle.

より高い密度を有するswarmerバンド、およびより低い密度のストーカー/ predivisionalセル帯域：同期は、典型的には、セルの二つのバンド（ 図1B）が得られる。効率的な同期を確実にするために共通のコントロールがOD 600を監視し、異なる細胞周期の時点でウエスタンブロットによりCTRAタンパク質のレベルを測定することを含む。 OD 600は、細胞周期（ 図2）</s...

Watch this Scientific Journal Video about Synchronization of Caulobacter Crescentus for Investigation of the Bacterial Cell Cycle at JoVE.com

Synchronization of Caulobacter Crescentus for Investigation of the Bacterial Cell Cycle

Synchronization of bacterial cells is essential for studies of the bacterial cell cycle and development. Caulobacter crescentus is synchronizable through density centrifugation allowing a rapid and powerful tool for studies of the bacterial cell cycle. Here we provide a detailed protocol for the synchronization of Caulobacter cells.

の同期<em&gt;カウロバクターCrescentus</em&gt;細菌細胞周期の調査のため

The cell cycle is important for growth, genome replication, and development in all cells.  In bacteria, studies of the cell cycle have focused largely on ...

synchronization-caulobacter-crescentus-for-investigation-bacterial

Research

JoVE Journal

Biology

Synchronization of Caulobacter Crescentus for Investigation of the Bacterial Cell Cycle

11.8K ビュー.  Stanford University School of Medicine. Synchronization of bacterial cells is essential for studies of the bacterial cell cycle and development. Caulobacter crescentus is synchronizable through density centrifugation allowing a rapid and powerful tool for studies of the bacterial cell cycle. Here we provide a detailed protocol for the synchronization of Caulobacter cells.

ビデオ: Synchronization of Caulobacter Crescentus for Investigation of the Bacterial Cell Cycle

Measuring the Bending Stiffness of Bacterial Cells Using an Optical Trap

We developed a protocol to measure the bending rigidity of filamentous rod-shaped bacteria. Forces are applied with an optical trap, a microscopic three-dimensional spring made of light that is formed when a high-intensity laser beam is focused to a very small spot by a microscope's objective lens. To bend a cell, we first bind live bacteria to a chemically-treated coverslip. As these cells grow, the middle of the cells remains bound to the coverslip but the growing ends are free of this restraint. By inducing filamentous growth with the drug cephalexin, we are able to identify cells in which one end of the cell was stuck to the surface while the other end remained unattached and susceptible to bending forces. A bending force is then applied with an optical trap by binding a polylysine-coated bead to the tip of a growing cell. Both the force and the displacement of the bead are recorded and the bending stiffness of the cell is the slope of this relationship.

We present a protocol for bending filamentous bacterial cells attached to a cover-slip surface with an optical trap to measure the cellular bending stiffness.

光トラップを用いた細菌細胞の曲げ剛性の測定

We developed a protocol to measure the bending rigidity of filamentous rod-shaped bacteria. Forces are applied with an optical trap, a microscopic ...

生物学

Monitoring Kinase and Phosphatase Activities Through the Cell Cycle by Ratiometric FRET

F&ouml;rster resonance energy transfer (FRET)-based reporters1 allow the assessment of endogenous kinase and phosphatase activities in living cells. Such probes typically consist of variants of CFP and YFP, intervened by a phosphorylatable sequence and a phospho-binding domain. Upon phosphorylation, the probe changes conformation, which results in a change of the distance or orientation between CFP and YFP, leading to a change in FRET efficiency (Fig 1). Several probes have been published during the last decade, monitoring the activity balance of multiple kinases and phosphatases, including reporters of PKA2, PKB3, PKC4, PKD5, ERK6, JNK7, Cdk18, Aurora B9 and Plk19. Given the modular design, additional probes are likely to emerge in the near future10. 
Progression through the cell cycle is affected by stress signaling pathways 11. Notably, the cell cycle is regulated differently during unperturbed growth compared to when cells are recovering from stress12.Time-lapse imaging of cells through the cell cycle therefore requires particular caution. This becomes a problem particularly when employing ratiometric imaging, since two images with a high signal to noise ratio are required to correctly interpret the results. Ratiometric FRET imaging of cell cycle dependent changes in kinase and phosphatase activities has predominately been restricted to sub-sections of the cell cycle8,9,13,14.
Here, we discuss a method to monitor FRET-based probes using ratiometric imaging throughout the human cell cycle. The method relies on equipment that is available to many researchers in life sciences and does not require expert knowledge of microscopy or image processing.

FRET-based reporters are increasingly used to monitor kinase and phosphatase activities in live cells. Here we describe a method on how to use FRET-based reporters to assess cell cycle-dependent changes in target phosphorylation.

FRETはレシオメトリックで細胞周期のキナーゼおよびホスファターゼの活動を監視

F&ouml;rster resonance energy transfer (FRET)-based reporters1 allow the assessment of endogenous kinase and phosphatase activities in living cells. Such ...

Analysis of Cell Cycle Position in Mammalian Cells

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of cell proliferation underlies the pathology of diseases like cancer. As such there is great need to be able to investigate cell proliferation and quantitate the proportion of cells in each phase of the cell cycle. It is also of vital importance to indistinguishably identify cells that are replicating their DNA within a larger population. Since a cell&prime;s decision to proliferate is made in the G1 phase immediately before initiating DNA synthesis and progressing through the rest of the cell cycle, detection of DNA synthesis at this stage allows for an unambiguous determination of the status of growth regulation in cell culture experiments.
DNA content in cells can be readily quantitated by flow cytometry of cells stained with propidium iodide, a fluorescent DNA intercalating dye. Similarly, active DNA synthesis can be quantitated by culturing cells in the presence of radioactive thymidine, harvesting the cells, and measuring the incorporation of radioactivity into an acid insoluble fraction. We have considerable expertise with cell cycle analysis and recommend a different approach. We Investigate cell proliferation using bromodeoxyuridine/fluorodeoxyuridine (abbreviated simply as BrdU) staining that detects the incorporation of these thymine analogs into recently synthesized DNA. Labeling and staining cells with BrdU, combined with total DNA staining by propidium iodide and analysis by flow cytometry1 offers the most accurate measure of cells in the various stages of the cell cycle. It is our preferred method because it combines the detection of active DNA synthesis, through antibody based staining of BrdU, with total DNA content from propidium iodide. This allows for the clear separation of cells in G1 from early S phase, or late S phase from G2/M. Furthermore, this approach can be utilized to investigate the effects of many different cell stimuli and pharmacologic agents on the regulation of progression through these different cell cycle phases.
In this report we describe methods for labeling and staining cultured cells, as well as their analysis by flow cytometry. We also include experimental examples of how this method can be used to measure the effects of growth inhibiting signals from cytokines such as TGF-&beta;1, and proliferative inhibitors such as the cyclin dependent kinase inhibitor, p27KIP1. We also include an alternate protocol that allows for the analysis of cell cycle position in a sub-population of cells within a larger culture5. In this case, we demonstrate how to detect a cell cycle arrest in cells transfected with the retinoblastoma gene even when greatly outnumbered by untransfected cells in the same culture. These examples illustrate the many ways that DNA staining and flow cytometry can be utilized and adapted to investigate fundamental questions of mammalian cell cycle control.

Determining the cell cycle position of a population of cells, or understanding how signals affect proliferation, can be readily measured by flow cytometry using this protocol. We report a simple experimental approach to staining cells and quantifying their position in the cell cycle.

哺乳動物細胞における細胞周期の位置の解析

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of ...

The ex vivo Isolated Skeletal Microvessel Preparation for Investigation of Vascular Reactivity

The isolated microvessel preparation is an ex vivo preparation that allows for examination of the different contributions of factors that control vessel diameter, and thus, perfusion resistance1-5. This is a classic experimental preparation that was, in large measure, initially described by Uchida et al.15 several decades ago. This initial description provided the basis for the techniques that was extensively modified and enhanced, primarily in the laboratory of Dr. Brian Duling at the University of Virginia6-8, and we present a current approach in the following pages. This preparation will specifically refer to the gracilis arteriole in a rat as the microvessel of choice, but the basic preparation can readily be applied to vessels isolated from nearly any other tissue or organ across species9-13. Mechanical (i.e., dimensional) changes in the isolated microvessels can easily be evaluated in response to a broad array of physiological (e.g., hypoxia, intravascular pressure, or shear) or pharmacological challenges, and can provide insight into mechanistic elements comprising integrated responses in an intact, although ex vivo, tissue. The significance of this method is that it allows for facile manipulation of the influences on the integrated regulation of microvessel diameter, while also allowing for the control of many of the contributions from other sources, including intravascular pressure (myogenic), autonomic innervation, hemodynamic (e.g., shear stress), endothelial dependent or independent stimuli, hormonal, and parenchymal influences, to provide a partial list. Under appropriate experimental conditions and with appropriate goals, this can serve as an advantage over in vivo or in situ tissue/organ preparations, which do not readily allow for the facile control of broader systemic variables. 
The major limitation of this preparation is essentially the consequence of its strengths. By definition, the behavior of these vessels is being studied under conditions where many of the most significant contributors to the regulation of vascular resistance have been removed, including neural, humoral, metabolic, etc. As such, the investigator is cautioned to avoid over-interpretation and extrapolation of the data that are collected utilizing this preparation. The other significant area of concern with regard to this preparation is that it can be very easy to damage cellular components such as the endothelial lining or the vascular smooth muscle, such that variable source of error can be introduced. It is strongly recommended that the individual investigator utilize appropriate measurements to ensure the quality of the preparation, both at the initiation of the experiment and periodically throughout the course of a protocol.

The ex vivo Isolated Skeletal Microvessel Preparation for Investigation of Vascular Reactivity

An ex vivo preparation is described for isolation of the largest gracilis muscle resistance arterioles for interrogation of both vascular responses to vasoactive stimuli and the assessment of basic structural properties via passive wall mechanics.

ザ ex vivoで</em血管反応性の調査のために>隔離された骨格微小血管の準備

The isolated  microvessel preparation is an ex vivo preparation that allows for examination of the different contributions of  factors that control vessel ...

Basic Caenorhabditis elegans Methods: Synchronization and Observation

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has since been used extensively as a model organism 1. C. elegans possesses key attributes such as simplicity, transparency and short life cycle that have made it a suitable experimental system for fundamental biological studies for many years 2. Discoveries in this nematode have broad implications because many cellular and molecular processes that control animal development are evolutionary conserved 3.
C. elegans life cycle goes through an embryonic stage and four larval stages before animals reach adulthood. Development can take 2 to 4 days depending on the temperature. In each of the stages several characteristic traits can be observed. The knowledge of its complete cell lineage 4,5 together with the deep annotation of its genome turn this nematode into a great model in fields as diverse as the neurobiology 6, aging 7,8, stem cell biology 9 and germ line biology 10. 
An additional feature that makes C. elegans an attractive model to work with is the possibility of obtaining populations of worms synchronized at a specific stage through a relatively easy protocol. The ease of maintaining and propagating this nematode added to the possibility of synchronization provide a powerful tool to obtain large amounts of worms, which can be used for a wide variety of small or high-throughput experiments such as RNAi screens, microarrays, massive sequencing, immunoblot or in situ hybridization, among others. 
Because of its transparency, C. elegans structures can be distinguished under the microscope using Differential Interference Contrast microscopy, also known as Nomarski microscopy. The use of a fluorescent DNA binder, DAPI (4',6-diamidino-2-phenylindole), for instance, can lead to the specific identification and localization of individual cells, as well as subcellular structures/defects associated to them.

Basic Caenorhabditis elegans Methods: Synchronization and Observation

The easiness of maintaining and propagating the nematode C. elegans make it a nice model organism to work with. The possibility of synchronizing worms allows the work with a significant amount of subjects at the same developmental stage, what facilitates the study of one particular process in many animals.

基本的な<em&gt;線虫Caenorhabditis elegans</em&gt;方法：同期との観測

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has ...

Temporal Tracking of Cell Cycle Progression Using Flow Cytometry without the Need for Synchronization

This protocol describes a method to permit the tracking of cells through the cell cycle without requiring the cells to be synchronized. Achieving cell synchronization can be difficult for many cell systems. Standard practice is to block cell cycle progression at a specific stage and then release the accumulated cells producing a wave of cells progressing through the cycle in unison. However, some cell types find this block toxic resulting in abnormal cell cycling, or even mass death. Bromodeoxyuridine (BrdU) uptake can be used to track the cell cycle stage of individual cells. Cells incorporate this synthetic thymidine analog, while synthesizing new DNA during S phase. By providing BrdU for a brief period it is possible to mark a pool of cells that were in S phase while the BrdU was present. These cells can then be tracked through the remainder of the cell cycle and into the next round of replication, permitting the duration of the cell cycle phases to be determined without the need to induce a potentially toxic cell cycle block. It is also possible to determine and correlate the expression of both internal and external proteins during subsequent stages of the cell cycle. These can be used to further refine the assignment of cell cycle stage or assess effects on other cellular functions such as checkpoint activation or cell death.

This protocol describes the use of bromodeoxyuridine (BrdU) uptake to permit the temporal tracking of cells that were in S phase at a specific point in time. Addition of DNA dyes and antibody labeling facilitates detailed analysis of the fate of the S phase cells at later times.

同期を必要とせずに、フローサイトメトリーを用いた細胞周期の進行の時間追跡

This protocol describes a method to permit the tracking of cells through the cell cycle without requiring the cells to be synchronized. Achieving cell ...

Efficient Mammalian Cell Expression and Single-step Purification of Extracellular Glycoproteins for Crystallization

Production of secreted mammalian proteins for structural and biophysical studies can be challenging, time intensive, and costly. Here described is a time and cost efficient protocol for secreted protein expression in mammalian cells and one step purification using nickel affinity chromatography. The system is based on large scale transient transfection of mammalian cells in suspension, which greatly decreases the time to produce protein, as it eliminates steps, such as developing expression viruses or generating stable expressing cell lines. This protocol utilizes cheap transfection agents, which can be easily made by simple chemical modification, or moderately priced transfection agents, which increase yield through increased transfection efficiency and decreased cytotoxicity. Careful monitoring and maintaining of media glucose levels increases protein yield. Controlling the maturation of native glycans at the expression step increases the final yield of properly folded and functional mammalian proteins, which are ideal properties to pursue X-ray crystallography. In some cases, single step purification produces protein of sufficient purity for crystallization, which is demonstrated here as an example case.

This is a quick, cost-efficient protocol for the production of secreted, glycosylated mammalian proteins and subsequent single-step purification with sufficient yields of homogenous protein for X-ray crystallography and other biophysical studies.

効率的な哺乳動物細胞発現および結晶化のための細胞外糖タンパク質のシングルステップの精製

Production of secreted mammalian proteins for structural and biophysical studies can be challenging, time intensive, and costly. Here described is a time ...

Measurement of Survival Time in Brachionus Rotifers: Synchronization of Maternal Conditions

Rotifers are microscopic cosmopolitan zooplankton used as models in ecotoxicological and aging studies due to their several advantages such as short lifespan, ease of culture, and parthenogenesis that enables clonal culture. However, caution is required when measuring their survival time as it is affected by maternal age and maternal feeding conditions. Here we provide a protocol for powerful and reproducible measurement of the survival time in Brachionus rotifers following a careful synchronization of culture conditions over several generations. Empirically, poor synchronization results in early mortality and a gradual decrease in survival rate, thus resulting in weak statistical power. Indeed, under such conditions, calorie restriction (CR) failed to significantly extend the lifespan of B. plicatilis although CR-induced longevity has been demonstrated with well-synchronized rotifer samples in past and present studies. This protocol is probably useful for other invertebrate models, including the fruitfly Drosophila melanogaster and the nematode Caenorhabditis elegans, because maternal age effects have also been reported in these species.

Measurement of Survival Time in Brachionus Rotifers: Synchronization of Maternal Conditions

Rotifers are microscopic zooplankton used as models in ecotoxicological and aging studies. Here we provide a protocol for powerful and reproducible measurement of survival time in Brachionus rotifers. Synchronization of culture conditions over several generations is of particular importance because maternal condition affects life history of offspring.

生存時間の測定で<em&gt; Brachionus</em&gt;ワムシ：母性条件の同期

Rotifers are microscopic cosmopolitan zooplankton used as models in ecotoxicological and aging studies due to their several advantages such as short ...

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

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

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

有糸分裂の多機能の細胞周期蛋白質の役割を研究する有糸分裂細胞同期と高分解能共焦点顕微鏡を組み合わせた

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

Live-Cell Imaging of the Life Cycle of Bacterial Predator Bdellovibrio bacteriovorus using Time-Lapse Fluorescence Microscopy

Bdellovibrio bacteriovorus is a small gram-negative, obligate predatory bacterium that kills other gram-negative bacteria, including harmful pathogens. Therefore, it is considered a living antibiotic. To apply B. bacteriovorus as a living antibiotic, it is first necessary to understand the major stages of its complex life cycle, particularly its proliferation inside prey. So far, it has been challenging to monitor successive stages of the predatory life cycle in real-time. Presented here is a comprehensive protocol for real-time imaging of the complete life cycle of B. bacteriovorus, especially during its growth inside the host. For this purpose, a system consisting of an agarose pad is used in combination with cell-imaging dishes, in which the predatory cells can move freely beneath the agarose pad while immobilized prey cells are able to form bdelloplasts. The application of a strain producing a fluorescently tagged &#946;-subunit of DNA polymerase III further allows chromosome replication to be monitored during the reproduction phase of the B. bacteriovorus life cycle.

Live-Cell Imaging of the Life Cycle of Bacterial Predator Bdellovibrio bacteriovorus using Time-Lapse Fluorescence Microscopy

Presented here is a protocol that describes monitoring of the complete life cycle of predatory bacterium Bdellovibrio bacteriovorus using time-lapse fluorescence microscopy in combination with an agarose pad and cell-imaging dishes.

タイムラプス蛍光顕微鏡を用いた細菌プレデター ブレロビトリオバクテリオボルス のライフサイクルの生細胞イメージング

Bdellovibrio bacteriovorus is a small gram-negative, obligate predatory bacterium that kills other gram-negative bacteria, including harmful pathogens. ...