Измерительная ячейка Кинетика цикла прогрессии с метаболическим Маркировка и проточной цитометрии
Summary
Отслеживание тонкие изменения в прогрессии и кинетики стадии клеточного цикла может быть достигнуто путем использования сочетания метаболических маркировки нуклеиновых кислот с BrdU и общая окраска геномной ДНК с помощью йодида пропидия. Этот метод позволяет избежать химического синхронизации велосипедного клеток, тем самым предотвращая внедрение неспецифического повреждения ДНК, которые, в свою очередь влияет на прогрессию клеточного цикла.
Abstract
Precise control of the initiation and subsequent progression through the various phases of the cell cycle are of paramount importance in proliferating cells. Cell cycle division is an integral part of growth and reproduction and deregulation of key cell cycle components have been implicated in the precipitating events of carcinogenesis 1,2. Molecular agents in anti-cancer therapies frequently target biological pathways responsible for the regulation and coordination of cell cycle division 3. Although cell cycle kinetics tend to vary according to cell type, the distribution of cells amongst the four stages of the cell cycle is rather consistent within a particular cell line due to the consistent pattern of mitogen and growth factor expression. Genotoxic events and other cellular stressors can result in a temporary block of cell cycle progression, resulting in arrest or a temporary pause in a particular cell cycle phase to allow for instigation of the appropriate response mechanism.
The ability to experimentally observe the behavior of a cell population with reference to their cell cycle progression stage is an important advance in cell biology. Common procedures such as mitotic shake off, differential centrifugation or flow cytometry-based sorting are used to isolate cells at specific stages of the cell cycle 4-6. These fractionated, cell cycle phase-enriched populations are then subjected to experimental treatments. Yield, purity and viability of the separated fractions can often be compromised using these physical separation methods. As well, the time lapse between separation of the cell populations and the start of experimental treatment, whereby the fractionated cells can progress from the selected cell cycle stage, can pose significant challenges in the successful implementation and interpretation of these experiments.
Other approaches to study cell cycle stages include the use of chemicals to synchronize cells. Treatment of cells with chemical inhibitors of key metabolic processes for each cell cycle stage are useful in blocking the progression of the cell cycle to the next stage. For example, the ribonucleotide reductase inhibitor hydroxyurea halts cells at the G1/S juncture by limiting the supply of deoxynucleotides, the building blocks of DNA. Other notable chemicals include treatment with aphidicolin, a polymerase alpha inhibitor for G1 arrest, treatment with colchicine and nocodazole, both of which interfere with mitotic spindle formation to halt cells in M phase and finally, treatment with the DNA chain terminator 5-fluorodeoxyridine to initiate S phase arrest 7-9. Treatment with these chemicals is an effective means of synchronizing an entire population of cells at a particular phase. With removal of the chemical, cells rejoin the cell cycle in unison. Treatment of the test agent following release from the cell cycle blocking chemical ensures that the drug response elicited is from a uniform, cell cycle stage-specific population. However, since many of the chemical synchronizers are known genotoxic compounds, teasing apart the participation of various response pathways (to the synchronizers vs. the test agents) is challenging.
Here we describe a metabolic labeling method for following a subpopulation of actively cycling cells through their progression from the DNA replication phase, through to the division and separation of their daughter cells. Coupled with flow cytometry quantification, this protocol enables for measurement of kinetic progression of the cell cycle in the absence of either mechanically- or chemically- induced cellular stresses commonly associated with other cell cycle synchronization methodologies 10. In the following sections we will discuss the methodology, as well as some of its applications in biomedical research.
Protocol
Discussion
Объединив проточной цитометрии с включением BrdU, у нас есть необходимые инструменты для изучения кинетики клеточного цикла. Отличительной особенностью BrdU функционировать как аналог тимидина то, что позволяет для содержания ДНК количественное велосипедные клетки. Включение BrdU в расту…
Divulgations
The authors have nothing to disclose.
Acknowledgements
Мы благодарим Энди Джонсон из Центра биомедицинских исследований в UBC для помощи в анализе FACS. Cancer Research финансирование в Вонг лаборатории обеспечивается канадской онкологическое общество Научно-исследовательский институт (рабочее грант № 019250), а также исследований реинвестирования средств факультет фармацевтических наук, UBC. JMYW поддерживается исследований кафедр и Канады Майкл Смит Фонд исследований в области здравоохранения программ развития карьеры.
Materials
| Reagent | Company | Catalogue Number | Comments |
| bromodeoxyuridine | Becton Dickinson | 55089 | |
| propidium iodide | Sigma | 287075 | 1mg/ml stock |
| FITC anti-BrdU | Becton Dickinson | 347583 | |
| sodium tetraborate | Fisher | S80172 | 0.1M, pH 8.5 |
| FACS Caliber | Becton Dickinson |
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