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. 대규모의 Synchrony - 웨스턴 블롯 (Western Blot), 마이크로 어레이 / RNA-SEQ 및 기타 재료 집중 분석 실험을위한 최적 <ol><li> 냉동 주식이나 접시에서 PYE 매체에서 28 ° C에서 흔들어 변형 NA1000의 5 ML의 O / N 문화를 성장. </li><li> M2G 25 ㎖ (표 1-2)에서 1 단계의 세포 0.5 ㎖를 접종하고, 문화는 0.5과 0.6 사이의 OD (600)에 도달 할 때까지 28 ° C에서 흔들. </li><li> M2G 1 L로 세포를 접종하고 28 ° C에서 흔들. </li><li> OD 600 0.6 0.5에 도달하면, 액체 장착 위상 현미경을 사용하여 세포 swarmer의 존재를 확인한다. 위상 현미경 유리 슬라이드에 세포의 1 μL가, 커버 슬립 커버 스팟 및 이미지. 인구 급속 수영 세포를 시각화하여 swarmer 세포의 존재를 확인한다. </li><li> JA-10 회에서 4 ° C에서 7 kxg에서 15 분 동안 세포를 스핀. </li><li> 뜨는을 취소하고 차가운 M2의 180 ml에 추가 (표 1-2)와 부드럽게 입술혈청 학적 피펫을 사용하여 모든 세포 uspend. 느슨하게 펠렛 세포를 폐기; 그들은 predivisional 및 스토킹 세포이다. </li><li> 차가운 콜로 이달 실리카 용액 60ml를 추가하고 잘 세포 현탁액을 혼합 (물론 셀에 추가하기 전에 콜로 이달 실리카 현탁액을 혼합해야합니다). </li><li> 여덟 30 ml의 튜브에 세포 현탁액을 붓고 JA-20 회에서 4 ° C에서 6.4 kxg에서 30 분 동안 회전. 참고 : 하나는 두 가지 밴드를 참조한다 스토킹 / predivisional 세포 TOP 밴드 (도 1b)에있는 동안 swarmer 대역 하위 밴드이다. </li><li> 조심스럽게 꺼 상단 밴드를 대기음 swarmer 밴드 (낮은 밴드) 위 ~ 1cm로 액체를 제거합니다. </li><li> 파스퇴르 피펫을 사용하여 (긴급),주의 깊게 swarmer 밴드를 제거하고 깨끗한 튜브에 배치합니다. JA-20 회에서 4 ° C에서 6.4 kxg에서 10 분 동안 콜로 이달 실리카, 최고 추위 M2와 튜브 오프 스핀을 씻어합니다. </li><li> 조심스럽게 상층 액을 제거하고를 재현 탁20 차가운 M2 ml의 JA-20 회에서 4 ° C에서 6.4 kxg에서 10 분 동안 스핀 세포. </li><li> 감기 M2의 30 ㎖ 중에 모든 펠릿을 재현 탁하고, 분광 광도계를 사용하여 OD (600)을 측정하고 차가운 M2 매체를 이용하여 빈. 세포 swarmer 확인하기 위해 위상 영상 1 μl를 저장; 세포 90-95 %가 swarmers해야한다. </li><li> JA-20 회에서 4 ° C에서 6.4 kxg에서 5 분 동안 세포를 스핀 다운. 28 ° C M2G 매체에 재현 탁 세포 600 ~ 0.3-0.4, 28 ℃에서 진탕 시작되도록. 참고 : 일반적인 수익률은 30 동기화되지 않은 세포의 1L에서 swarmer 세포 배양의 60ML 사이. </li><li> 시점 (야생형 문화를 나누는 약 135-140분 소요됩니다) 8,9를 복용 시작합니다. 각 시점에서, OD (600)을 측정한다. 분할 후 OD (600)는 약 2 배의 초기 OD 600 있는지 확인합니다. </li><li> 웨스턴 블롯 또는 유전자 발현 분석을 위해, 용돈의 1 ㎖ 씩 제거원하는 시점에 lture는, 빠르게, 30 초 동안 탁상 원심 분리기에서 최대 속도로 스핀 다운 가만히 따르다 또는 매체를 대기음, 플래시는 액체 질소에서 세포 펠렛을 동결. 다운 스트림 분석 할 때까지 -80 ° C에서 세포를 저장합니다. </li></ol> 2. 작은 규모의 Synchrony - 현미경을위한 최적 <ol><li> 냉동 주식이나 접시에서 5 ML의 O / N 문화 M2G 28 ° C에서 진탕 성장한다. </li><li> M2G 15 ㎖ (표 1-2)에 희석 중반 로그 (OD = 0.5 ~ 0.6 600)까지 성장한다. </li><li> 1 ml의 차가운 M2 (표 1-2)과 2 ML의 microcentrifuge 관에 전송에서 JA-20 회에서 4 ° C에서 10 분 6.4 kxg에 스핀, 그리고에 resuspend. </li><li> 뜨는을 대기음, 펠릿 세포 microcentrifuge 관에서 3 분 15 kxg에 스핀, 감기 M2의 900 μL에 얼음에 펠릿 및 재현 탁했습니다. </li><li> 마이크에서 4 ° C에서 15 kxg에서 20 분 동안 콜로 이달 실리카 스핀 코팅 감기 PVP의 900 μl를 추가rocentrifuge 튜브. </li><li> (긴급) 대기음은 최고에게 스토킹 / predivisional 세포 밴드를 피펫하고 새로운 microcentrifuge 관에 밴드 swarmer 바닥을 수집하거나. </li><li> 3 분 15 kxg에서 원심 분리 동안 감기 M2의 1 ml의 swarmer 세포를 두 번 씻으십시오. </li><li> 최종 스핀 앞서, 냉장 1ml의 유리 시험관에 세포 이동 및 M2의 블랭크에 비해 셀 OD (600)을 측정한다. </li><li> 0.3 사이의 OD에서 28 ° C M2G에 최종 세포 펠렛을 재현 탁 - 0.4, 28 ℃에서 / 롤 세포를 흔들어. 참고 : 일반적인 수익률이 swarmer 세포 배양의 2 내지 4 ml의입니다. </li><li> 이미징을위한 M2G 아가로 오스 패드 상에 세포의 원하는 시점 장소 1 μL에서 현미경 실험하십시오. </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_...

박테리아 세포 사이클은 게놈의 복제와 딸 세포 분열의 양을 제어한다. 항생제 내성은 공중 보건에 대한 위협도 갈수록 증가로 중요한 것은, 세균 세포주기 항생제 개발을위한 미개척 목표를 제시한다. 세균 Caulobacter crescentus의, 각 셀은 서로 다른주기 운명 (도 1a)의 1,2- 개의 딸 세포를 수득 비대칭 분할 리드. 한 딸 셀은 편모를 상속하고 다른 딸이 줄기를 상속 고착 동안 운동성이있다. 통합 유전자 회로는 전사 조절, 인산화 신호 전달 및 조절 단백질 분해 (3)에 의한 세포주기 진행과 세포의 운명을 제어합니다. 또한, 염색체 복제 및 동시 분리 수율 딸 세포는 염색체 4의 단 하나의 복사본을 포함하고있다. 중요한 것은, 이러한 두 가지 종류의 세포는 급속 collo 의해 분리 될 수있다높은 수율 (도 1b)와 인구의 나머지 swarmer 세포의 분리를 허용하는 동기화 가능한 균주의 NA1000 5-7 idal 실리카 입자 밀도 원심. 세포 swarmer 절연 한 후 비대칭 세포 분열을 통해 동 기적으로 진행합니다. 여기서 우리는 세부 프로토콜은 Caulobacter 변형 NA1000를 동기화에 사용됩니다. 우리는 프로토콜과의 크고 작은 규모 모두 동기화에 대한 일반적인 문제 해결 팁을 제공합니다. 이 실험 절차는 Caulobacter의 세포주기 및 세포 운명의 시공간 제어를 심문 할 수있는 강력한 도구를 제공합니다.

<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.

높은 밀도가 swarmer 밴드, 낮은 밀도의 스토킹 / predivisional 세포 밴드 : 동기화는 일반적으로 세포의 두 밴드 (그림 1B)를 산출한다. 효율적인 동기화 공통 컨트롤 OD 600를 모니터링하고 뚜렷한 세포주기 시점에서 웨스턴 블롯 CTRA 단백질의 수준을 측정 포함되도록한다. OD (600)는 세포주기의 과정 (도 2) 동안 약 2 배 증가한다. 세포주기 마스터 레귤레이터 CTRA ...

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.

의 동기화<em&gt; Caulobacter Crescentus</em&gt; 세균 세포주기의 조사에 대한

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 ...

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Biology

Synchronization of Caulobacter Crescentus for Investigation of the Bacterial Cell Cycle

11.8K 조회수.  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.

비디오: 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.

광학 트랩을 사용하여 박테리아 세포의 굽힘 강성을 측정

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

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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.

안달 Ratiometric하여 세포주기를 통해 키나제와 인산 가수 분해 효소 활동을 모니터링

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.

포유 동물 세포의 세포주기의 위치 분석

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.

예 생체내</em혈관 반응성 조사를위한> 절연 골격 Microvessel 준비

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.

기본<em&gt; Caenorhabditis elegans</em&gt; 방법 : 동기화 및 전망

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.

동기화를 필요로하지 않고 유동 세포 계측법을 사용하여 세포주기 진행의 시간적 추적

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.

효율적인 포유 동물 세포 발현 및 결정화에 대한 세포 외 당 단백질의 단일 단계 정화

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.

생존 시간의 측정에<em&gt; Brachionus</em&gt; 로티퍼 : 산모 조건의 동기화

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.

유사 분열 동안 다기능 세포 주기 단백질의 역할을 연구 하기 Mitotic 세포 동기화와 고해상도 Confocal 현미경 검사 법

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.

시간 경과 형광 현미경 검사를 사용하여 세균 육식 동물 Bdellovibrio 박테리오보루스의 라이프 사이클의 라이브 세포 이미징

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