Name: measuring cell cycle progression kinetics with metabolic labeling and flow cytometry
Uploaded: 2012-05-22T13:00:00
Description: Watch this Scientific Journal Video about Measuring Cell Cycle Progression Kinetics with Metabolic Labeling and Flow Cytometry at JoVE.com

हम UBC पर बायोमेडिकल रिसर्च सेंटर के एंडी जॉनसन FACS विश्लेषण के साथ सहायता के लिए धन्यवाद. कैंसर वाँग प्रयोगशाला में अनुसंधान अनुदान कनाडा के कैंसर सोसायटी अनुसंधान संस्थान (ऑपरेटिंग अनुदान 019,250) और अनुसंधान, औषधि विज्ञान, UBC के संकाय के पुनर्निवेश कोष से प्रदान की जाती है. JMYW कनाडा अनुसंधान अध्यक्षों और स्वास्थ्य अनुसंधान कैरियर विकास कार्यक्रमों के लिए माइकल स्मिथ फाउंडेशन द्वारा समर्थित है.

1. सेल तैयार <img src="/files/ftp_upload/4045/4045fig0.jpg" alt="0 चित्रा" /> <ol><li> ?...

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	<li>Musgrove, E. A., Caldon, C. E., Barraclough, J., Stone, A., Sutherland, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cyclin+D+as+a+therapeutic+target+in+cancer.">Cyclin D as a therapeutic target in cancer.</a> Nat. Rev. Cancer. 11, 558-572 (2011).</li><li>Molchadsky, A., Rivlin, N., Brosh, R., Rotter, V., Sarig, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=p53+is+balancing+development,+differentiation+and+de-differentiation+to+assure+cancer+prevention.">p53 is balancing development, differentiation and de-differentiation to assure cancer prevention.</a> Carcinogenesis. 31, 1501-1508 (2010).</li><li>Dickson, M. A., Schwartz, G. 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V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synchronization+of+cell+populations+in+G1/S+and+G2/M+phases+of+the+cell+cycle.">Synchronization of cell populations in G1/S and G2/M phases of the cell cycle.</a> Methods Mol. Biol. 296, 157-166 (2005).</li><li>Pedrali-Noy, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synchronization+of+HeLa+cell+cultures+by+inhibition+of+DNA+polymerase+alpha+with+aphidicolin.">Synchronization of HeLa cell cultures by inhibition of DNA polymerase alpha with aphidicolin.</a> Nucleic Acids Res. 8, 377-387 (1980).</li><li>Merrill, G. 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BrdU समावेश के साथ प्रवाह cytometry के संयोजन के द्वारा, हम सेल चक्र कैनेटीक्स का अध्ययन करने के लिए आवश्यक उपकरण हैं. BrdU thymidine एनालॉग के रूप में समारोह के विशिष्ट संपत्ति है क्या एक साइकिल कोशिका के डीएनए सामग्री ?...

<table border="1"><tbody><tr><td> अभिकर्मक </td><td> कंपनी </td><td> सूचीपत्र संख्या </td><td> टिप्पणियाँ </td></tr><tr><td> bromodeoxyuridine </td><td> Becton डिकिंसन </td><td> ५५,०८९ </td><td></td></tr><tr><td> propidium आयोडाइड </td><td> सिग्मा </td><td> 287,075 </td><td> 1mg/ml स्टॉक </td></tr><tr><td> विरोधी BrdU FITC </td><td> Becton डिकिंसन </td><td> +३४७५८३ </td><td></td></tr><tr><td> सोडियम tetraborate </td><td> मछुआ </td><td> S80172 </td><td> 0.1M, 8.5 पीएच </td></tr><tr><td> FACS कैलिबर </td><td> Becton डिकिंसन </td><td></td><td></td></tr></tbody></table>

measuring cell cycle progression kinetics with metabolic labeling and flow cytometry

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.

Watch this Scientific Journal Video about Measuring Cell Cycle Progression Kinetics with Metabolic Labeling and Flow Cytometry at JoVE.com

Measuring Cell Cycle Progression Kinetics with Metabolic Labeling and Flow Cytometry

ट्रैकिंग और सेल चक्र के चरणों की प्रगति कैनेटीक्स में सूक्ष्म परिवर्तन nucleic एसिड के Propidium आयोडाइड के माध्यम से BrdU और कुल जीनोमिक डीएनए धुंधला के साथ चयापचय लेबलिंग का एक संयोजन का उपयोग करके पूरा किया जा सकता है. इस विधि साइकिल कोशिकाओं के रासायनिक तुल्यकालन की जरूरत से बचा जाता है, जिससे गैर विशिष्ट डीएनए की क्षति, जो बारी में कोशिका चक्र प्रगति को प्रभावित करता है की शुरूआत को रोकने.

मेटाबोलिक लेबल और फ्लो के साथ कोशिका चक्र प्रगति कैनेटीक्स मापने

Precise control of the initiation and subsequent progression through the various phases of the cell cycle are of paramount importance in proliferating ...

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