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><tbody><tr><td>PVP Coated Colloidal Silica (Percoll)</td><td>Sigma-Aldrich</td><td>P4937</td><td></td></tr><tr><td>Colloidal Silica (Ludox AS-40)</td><td>Sigma-Aldrich</td><td>420840</td><td></td></tr><tr><td>JA10 Rotor</td><td>Beckman-Coulter</td><td>369687</td><td></td></tr><tr><td>JA20 Rotor</td><td>Beckman-Coulter</td><td>334831</td><td></td></tr><tr><td>Ferrous Sulfate Chelate Solution</td><td>Sigma-Aldrich</td><td>F0518</td><td></td></tr><tr><td>30 ml Centrifuge Tubes</td><td>Corning</td><td>8445</td><td></td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td><td>EMD</td><td>SX0720-1</td><td></td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>VWR</td><td>BDH9268-500G</td><td></td></tr><tr><td>NH&lt;sub&gt;4&lt;/sub&gt;Cl</td><td>Amresco</td><td>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 ...

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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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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

Studying Cell Cycle-regulated Gene Expression by Two Complementary Cell Synchronization Protocols

The gene expression program of the cell cycle represents a critical step for understanding cell cycle-dependent processes and their role in diseases such as cancer. Cell cycle-regulated gene expression analysis depends on cell synchronization into specific phases. Here we describe a method utilizing two complementary synchronization protocols that is commonly used for studying periodic variation of gene expression during the cell cycle. Both procedures are based on transiently blocking the cell cycle in one defined point. The synchronization protocol by hydroxyurea (HU) treatment leads to cellular arrest in late G1/early S phase, and release from HU-mediated arrest provides a cellular population uniformly progressing through S and G2/M. The synchronization protocol by thymidine and nocodazole (Thy-Noc) treatment blocks cells in early mitosis, and release from Thy-Noc mediated arrest provides a synchronized cellular population suitable for G1 phase and S phase-entry studies. Application of both procedures requires monitoring of the cell cycle distribution profiles, which is typically performed after propidium iodide (PI) staining of the cells and flow cytometry-mediated analysis of DNA content. We show that the combined use of two synchronization protocols is a robust approach to clearly determine the transcriptional profiles of genes that are differentially regulated in the cell cycle (i.e. E2F1 and E2F7), and consequently to have a better understanding of their role in cell cycle processes. Furthermore, we show that this approach is useful for the study of mechanisms underlying drug-based therapies (i.e. mitomycin C, an anticancer agent), because it allows to discriminate genes that are responsive to the genotoxic agent from those solely affected by cell cycle perturbations imposed by the agent.

We report two cell synchronization protocols that provide a context for studying events related to specific phases of the cell cycle. We show that this approach is useful for analyzing the regulation of specific genes in an unperturbed cell cycle or upon exposure to agents affecting the cell cycle.

두 개의 상보적인 세포 동기화 프로토콜에 의한 세포주기 조절 유전자 발굴 연구

The gene expression program of the cell cycle represents a critical step for understanding cell cycle-dependent processes and their role in diseases such ...

연구

유전학

Live Cell Imaging of Bacillus subtilis and Streptococcus pneumoniae using Automated Time-lapse Microscopy

During the last few years scientists became increasingly aware that average data obtained from microbial population based experiments are not representative of the behavior, status or phenotype of single cells. Due to this new insight the number of single cell studies rises continuously (for recent reviews see 1,2,3). However, many of the single cell techniques applied do not allow monitoring the development and behavior of one specific single cell in time (e.g. flow cytometry or standard microscopy).
Here, we provide a detailed description of a microscopy method used in several recent studies 4, 5, 6, 7, which allows following and recording (fluorescence of) individual bacterial cells of Bacillus subtilis and Streptococcus pneumoniae through growth and division for many generations. The resulting movies can be used to construct phylogenetic lineage trees by tracing back the history of a single cell within a population that originated from one common ancestor. This time-lapse fluorescence microscopy method cannot only be used to investigate growth, division and differentiation of individual cells, but also to analyze the effect of cell history and ancestry on specific cellular behavior. Furthermore, time-lapse microscopy is ideally suited to examine gene expression dynamics and protein localization during the bacterial cell cycle. The method explains how to prepare the bacterial cells and construct the microscope slide to enable the outgrowth of single cells into a microcolony. In short, single cells are spotted on a semi-solid surface consisting of growth medium supplemented with agarose on which they grow and divide under a fluorescence microscope within a temperature controlled environmental chamber. Images are captured at specific intervals and are later analyzed using the open source software ImageJ.

Live Cell Imaging of Bacillus subtilis and Streptococcus pneumoniae using Automated Time-lapse Microscopy

This protocol provides a step-by-step procedure to monitor single cell behavior of different bacteria in time using automated fluorescence time-lapse microscopy. Furthermore, we provide guidelines how to analyze the microscopy images.

라이브 셀 이미징 바실러스 subtilis과 연쇄상 구균 pneumoniae 자동 시간 저속 현미경을 사용하여

During the last few years scientists became increasingly aware that average data obtained from microbial population based experiments are not ...

면역학 및 감염병학

Fluorescence Live-cell Imaging of the Complete Vegetative Cell Cycle of the Slow-growing Social Bacterium Myxococcus xanthus

Fluorescence live-cell imaging of bacterial cells is a key method in the analysis of the spatial and temporal dynamics of proteins and chromosomes underlying central cell cycle events. However, imaging of these molecules in slow-growing bacteria represents a challenge due to photobleaching of fluorophores and phototoxicity during image acquisition. Here, we describe a simple protocol to circumvent these limitations in the case of Myxococcus xanthus (which has a generation time of 4 - 6 h). To this end, M. xanthus cells are grown on a thick nutrient-containing agar pad in a temperature-controlled humid environment. Under these conditions, we determine the doubling time of individual cells by following the growth of single cells. Moreover, key cellular processes such as chromosome segregation and cell division can be imaged by fluorescence live-cell imaging of cells containing relevant fluorescently labeled marker proteins such as ParB-YFP, FtsZ-GFP, and mCherry-PomX over multiple cell cycles. Subsequently, the acquired images are processed to generate montages and/or movies.

Fluorescence Live-cell Imaging of the Complete Vegetative Cell Cycle of the Slow-growing Social Bacterium Myxococcus xanthus

Bacterial cells are spatially highly organized. To follow this organization over time in slow growing Myxococcus xanthus cells, a set-up for fluorescence live-cell imaging with high spatiotemporal resolution over several generations was developed. Using this method, spatiotemporal dynamics of important proteins for chromosome segregation and cell division could be determined.

형광 라이브 셀 이미징의 완전 한 식물 세포의 주기 느린 성장 사회 박테리아 Myxococcus xanthus

Fluorescence live-cell imaging of bacterial cells is a key method in the analysis of the spatial and temporal dynamics of proteins and chromosomes ...

생화학

Continuous Measurement of Biological Noise in Escherichia Coli Using Time-lapse Microscopy

The protocol developed here offers a tool to enable computer tracking of Escherichia coli division and fluorescent levels over several hours. The process starts by screening for colonies that survive on minimal media, assuming that only Escherichia coli harboring the correct plasmid will be able to thrive in the specific conditions. Since the process of building large genetic circuits, requiring the assembly of many DNA parts, is challenging, circuit components are often distributed between multiple plasmids at different copy numbers requiring the use of several antibiotics. Mutations in the plasmid can destroy transcription of the antibiotic resistance genes and interject with resources management in the cell leading to necrosis. The selected colony is set on a glass-bottom Petri dish and a few focus planes are selected for microscopy tracking in both bright field and fluorescent domains. The protocol maintains the image focus for more than 12 hours under initial conditions that cannot be regulated, creating a few difficulties. For example, dead cells start to accumulate in the lenses' field of focus after a few hours of imaging, which causes toxins to buildup and the signal to blur and decay. Depletion of nutrients introduces new metabolic processes and hinder the desired response of the circuit. The experiment's temperature lowers the effectivity of inducers and antibiotics, which can further damage the reliability of the signal. The minimal media gel shrinks and dries, and as a result the optical focus changes over time. We developed this method to overcome these challenges in Escherichia coli, similar to previous works developing analogous methods for other micro-organisms. In addition, this method offers an algorithm to quantify the total stochastic noise in unaltered and altered cells, finding that the results are consistent with flow analyzer predictions as shown by a similar coefficient of variation (CV).

Continuous Measurement of Biological Noise in Escherichia Coli Using Time-lapse Microscopy

This work presents a microscopy method that allows live imaging of a single cell of Escherichia coli for analysis and quantification of the stochastic behavior of synthetic gene circuits.

시간 경과 현미경 검사를 사용하여 대장균에서 생물학적 소음의 지속적인 측정

The protocol developed here offers a tool to enable computer tracking of Escherichia coli division and fluorescent levels over several hours. The process ...

생체공학

Live-Cell Fluorescence Microscopy to Investigate Subcellular Protein Localization and Cell Morphology Changes in Bacteria

Investigations of factors influencing cell division and cell shape in bacteria are commonly performed in conjunction with high-resolution fluorescence microscopy as observations made at a population level may not truly reflect what occurs at a single cell level. Live-cell timelapse microscopy allows investigators to monitor the changes in cell division or cell morphology which provide valuable insights regarding subcellular localization of proteins and timing of gene expression, as it happens, to potentially aid in answering important biological questions. Here, we describe our protocol to monitor phenotypic changes in Bacillus subtilis and Staphylococcus aureus using a high-resolution deconvolution microscope. The objective of this report is to provide a simple and clear protocol that can be adopted by other investigators interested in conducting fluorescence microscopy experiments to study different biological processes in bacteria as well as other organisms.

This article provides a step-by-step guide to investigate protein subcellular localization dynamics and to monitor morphological changes using high-resolution fluorescence microscopy in Bacillus subtilis and Staphylococcus aureus.

박테리아의 세포 외 단백질 국소화 및 세포 형태 변화를 조사하는 살아있는 세포 형광 현미경 검사법

Investigations of factors influencing cell division and cell shape in bacteria are commonly performed in conjunction with high-resolution fluorescence ...

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 ...

생물학

Live Cell Fluorescence Microscopy to Observe Essential Processes During Microbial Cell Growth

Core cellular processes such as DNA replication and segregation, protein synthesis, cell wall biosynthesis, and cell division rely on the function of proteins which are essential for bacterial survival. A series of target-specific dyes can be used as probes to better understand these processes. Staining with lipophilic dyes enables the observation of membrane structure, visualization of lipid microdomains, and detection of membrane blebs. Use of fluorescent-d-amino acids (FDAAs) to probe the sites of peptidoglycan biosynthesis can indicate potential defects in cell wall biogenesis or cell growth patterning. Finally, nucleic acid stains can indicate possible defects in DNA replication or chromosome segregation. Cyanine DNA stains label living cells and are suitable for time-lapse microscopy enabling real-time observations of nucleoid morphology during cell growth. Protocols for cell labeling can be applied to protein depletion mutants to identify defects in membrane structure, cell wall biogenesis, or chromosome segregation. Furthermore, time-lapse microscopy can be used to monitor morphological changes as an essential protein is removed and can provide additional insights into protein function. For example, the depletion of essential cell division proteins results in filamentation or branching, whereas the depletion of cell growth proteins may cause cells to become shorter or rounder. Here, protocols for cell growth, target-specific labeling, and time-lapse microscopy are provided for the bacterial plant pathogen Agrobacterium tumefaciens. Together, target-specific dyes and time-lapse microscopy enable characterization of essential processes in A. tumefaciens. Finally, the protocols provided can be readily modified to probe essential processes in other bacteria.

Understanding the function of essential processes in bacteria is challenging. Fluorescence microscopy with target-specific dyes can provide key insights into microbial cell growth and cell cycle progression. Here, Agrobacterium tumefaciens is used as a model bacterium to highlight methods for live cell imaging for characterization of essential processes.

라이브 세포 형광 현미경 검사 법 미생물 세포 성장 하는 동안 필수 과정을 관찰 하

Core cellular processes such as DNA replication and segregation, protein synthesis, cell wall biosynthesis, and cell division rely on the function of ...

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 ...

Single-cell Microfluidic Analysis of Bacillus subtilis

Microfluidic technology overcomes many of the limitations to traditional analytical methods in microbiology. Unlike bulk-culture methods, it offers single-cell resolution and long observation times spanning hundreds of generations; unlike agarose pad-based microscopy, it has uniform growth conditions that can be tightly controlled. Because the continuous flow of growth medium isolates the cells in a microfluidic device from unpredictable variations in the local chemical environment caused by cell growth and metabolism, authentic changes in gene expression and cell growth in response to specific stimuli can be more confidently observed. Bacillus subtilis is used here as a model bacterial species to demonstrate a &#34;mother machine&#34;-type method for cellular analysis. We show how to construct and plumb a microfluidic device, load it with cells, initiate microscopic imaging, and expose cells to a stimulus by switching from one growth medium to another. A stress-responsive reporter is used as an example to reveal the type of data that may be obtained by this method. We also briefly discuss further applications of this method for other types of experiments, such as analysis of bacterial sporulation.

Single-cell Microfluidic Analysis of Bacillus subtilis

We present a method for the microfluidic analysis of individual bacterial cell lineages using Bacillus subtilis as an example. The method overcomes shortcomings of traditional analytical methods in microbiology by allowing observation of hundreds of cell generations under tightly controllable and uniform growth conditions.

새 균의 subtilis 의 단일 셀 미세 분석

Microfluidic technology overcomes many of the limitations to traditional analytical methods in microbiology. Unlike bulk-culture methods, it offers ...

Laser Micro-Irradiation to Study DNA Recruitment During S Phase

DNA damage repair maintains the genetic integrity of cells in a highly reactive environment. Cells may accumulate various types of DNA damage due to both endogenous and exogenous sources such as metabolic activities or UV radiation. Without DNA repair, the cell's genetic code becomes compromised, undermining the structures and functions of proteins and potentially causing disease. 
Understanding the spatiotemporal dynamics of the different DNA repair pathways in various cell cycle phases is crucial in the field of DNA damage repair. Current fluorescent microscopy techniques provide great tools to measure the recruitment kinetics of different repair proteins after DNA damage induction. DNA synthesis during the S phase of the cell cycle is a peculiar point in cell fate regarding DNA repair. It provides a unique window to screen the entire genome for mistakes. At the same time, DNA synthesis errors also pose a threat to DNA integrity that is not encountered in non-dividing cells. Therefore, DNA repair processes differ significantly in S phase as compared to other phases of the cell cycle, and those differences are poorly understood. 
The following protocol describes the preparation of cell lines and the measurement of dynamics of DNA repair proteins in S phase at locally induced DNA damage sites, using a laser-scanning confocal microscope equipped with a 405 nm laser line. Tagged PCNA (with mPlum) is used as a cell cycle marker combined with an AcGFP-labeled repair protein of interest (i.e., EXO1b) to measure the DNA damage recruitment in S phase.

This protocol describes a non-invasive method to efficiently identify S-phase cells for downstream microscopy studies, such as measuring DNA repair protein recruitment by laser micro-irradiation.

S 단계 동안 DNA 모집을 연구하는 레이저 미세 조사

DNA damage repair maintains the genetic integrity of cells in a highly reactive environment. Cells may accumulate various types of DNA damage due to both ...

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