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

We thank Andy Johnson of the Biomedical Research Center at UBC for assistance with FACS analysis. Cancer Research Funding in the Wong Laboratory is provided by the Canadian Cancer Society Research Institute (operating grant #019250) and from Research Reinvestment funds of the Faculty of Pharmaceutical Sciences, UBC. JMYW is supported by the Canada Research Chairs and the Michael Smith Foundation for Health Research Career Development programs.

1. Cell Preparation
<img src="/files/ftp_upload/4045/4045fig0.jpg" alt="Figure 0" /> 
<ol>
 <li> Plate cells t...

<ol>
	<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. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+cell-cycle+inhibitors+for+cancer+therapy.">Development of cell-cycle inhibitors for cancer therapy.</a> Curr. Oncol. 16, 36-43 (2009).</li><li>Banfalvi, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+cycle+synchronization+of+animal+cells+and+nuclei+by+centrifugal+elutriation.">Cell cycle synchronization of animal cells and nuclei by centrifugal elutriation.</a> Nat. Protoc. 3, 663-673 (2008).</li><li>van Opstal, A., Boonstra, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibitors+of+phosphatidylinositol+3-kinase+activity+prevent+cell+cycle+progression+and+induce+apoptosis+at+the+M/G1+transition+in+CHO+cells.">Inhibitors of phosphatidylinositol 3-kinase activity prevent cell cycle progression and induce apoptosis at the M/G1 transition in CHO cells.</a> Cell Mol. Life Sci. 63, 220-228 (2006).</li><li>Gasnereau, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometry+to+sort+mammalian+cells+in+cytokinesis.">Flow cytometry to sort mammalian cells in cytokinesis.</a> Cytometry. A. 71, 1-7 (2007).</li><li>Harper, J. 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. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+synchronization.">Cell synchronization.</a> Methods Cell Biol. 57, 229-249 (1998).</li><li>Fleisig, H. B., Wong, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Telomerase+promotes+efficient+cell+cycle+kinetics+and+confers+growth+advantage+to+telomerase-negative+transformed+human+cells.">Telomerase promotes efficient cell cycle kinetics and confers growth advantage to telomerase-negative transformed human cells.</a> Oncogene. , (2011).</li><li>Cai, D., Byth, K. F., Shapiro, G. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AZ703,+an+imidazo[1,2-a]pyridine+inhibitor+of+cyclin-dependent+kinases+1+and+2,+induces+E2F-1-dependent+apoptosis+enhanced+by+depletion+of+cyclin-dependent+kinase+9.">AZ703, an imidazo[1,2-a]pyridine inhibitor of cyclin-dependent kinases 1 and 2, induces E2F-1-dependent apoptosis enhanced by depletion of cyclin-dependent kinase 9.</a> Cancer Res. 66, 435-444 (2006).</li><li>Pozarowski, P., Darzynkiewicz, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+cell+cycle+by+flow+cytometry.">Analysis of cell cycle by flow cytometry.</a> Methods Mol. Biol. 281, 301-311 (2004).</li><li>Sherwood, S. W., Rush, D. F., Kung, A. L., Schimke, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cyclin+B1+expression+in+HeLa+S3+cells+studied+by+flow+cytometry.">Cyclin B1 expression in HeLa S3 cells studied by flow cytometry.</a> Exp. Cell Res. 211, 275-281 (1994).</li></ol>

By combining flow cytometry with BrdU incorporation, we have the necessary tools to study cell cycle kinetics. The distinctive property of BrdU to function as a thymidine analogue is what allows for DNA content quantification of a cycling cell. The incorporation of BrdU into a growing daughter DNA strand during the synthesis phase of the cell cycle is what allows one to follow a subpopulation of cycling cells through the replication of DNA in S phase, to a period of growth at G2 and ultimately to cell division. Daught...

<table border="1">
<tbody>
 <tr>
 <td>Reagent</td>
 <td>Company</td>
 <td>Catalogue Number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>bromodeoxyuridine</td>
 <td>Becton Dickinson</td>
 <td>55089</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>propidium iodide</td>
 <td>Sigma</td>
 <td>287075</td>
 <td>1mg/ml stock</td>
 </tr>
 <tr>
 <td>FITC anti-BrdU</td>
 <td>Becton Dickinson</td>
 <td>347583</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>sodium tetraborate</td>
 <td>Fisher</td>
 <td>S80172</td>
 <td>0.1M, pH 8.5</td>
 </tr>
 <tr>
 <td>FACS Caliber</td>
 <td>Becton Dickinson</td>
 <td>&nbsp;</td>
 <td>&nbsp;</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 (Video) | JoVE

Tracking subtle changes in the progression and kinetics of cell cycle stages can be accomplished by use of a combination of metabolic labeling of nucleic acids with BrdU and total genomic DNA staining via Propidium Iodide. This method avoids the need of chemical synchronization of cycling cells, thereby preventing the introduction of non-specific DNA damage, which in turn affects cell cycle progression.

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 ...

Research

JoVE Journal

Biology

Measuring Exocytosis in Neurons Using FM Labeling

Measuring Exocytosis in Neurons Using FM Labeling (Video) | JoVE

The ability to measure the kinetics of vesicle release can help provide insight into some of the basics of neurotransmission.  Here we used real-time ...

Studying Membrane Biogenesis with a Luciferase-Based Reporter Gene Assay

Studying Membrane Biogenesis with a Luciferase-Based Reporter Gene Assay (Video) | JoVE

To study the coordination of different lipid synthesis pathways during membrane biogenesis it is useful to work with an experimental system where membrane ...

Flow Cytometry Purification of Mouse Meiotic Cells

Flow Cytometry Purification of Mouse Meiotic Cells (Video) | JoVE

The heterogeneous nature of cell types in the testis and the absence of meiotic cell culture models have been significant hurdles to the study of the ...

Flow Cytometry-based Purification of S. cerevisiae Zygotes

Flow Cytometry-based Purification of S. cerevisiae Zygotes (Video) | JoVE

Zygotes are essential intermediates between haploid and diploid states in the life cycle of many organisms, including yeast (Figure 1) 1. S. cerevisiae ...

Optimized Staining and Proliferation Modeling Methods for Cell Division Monitoring using Cell Tracking Dyes

Optimized Staining and Proliferation Modeling Methods for Cell Division Monitoring using Cell Tracking Dyes (Video) | JoVE

Fluorescent cell tracking dyes, in combination with flow and image cytometry, are powerful tools with which to study the interactions and fates of ...

Measuring Intracellular Ca2+ Changes in Human Sperm using Four Techniques: Conventional Fluorometry, Stopped Flow Fluorometry, Flow Cytometry and Single Cell Imaging

Measuring Intracellular Ca2+ Changes in Human Sperm using Four Techniques: Conventional Fluorometry, Stopped Flow Fluorometry, Flow Cytometry and Single Cell Imaging (Video) | JoVE

Spermatozoa  are male reproductive cells especially designed to reach, recognize and fuse  with the egg. To perform these tasks, sperm cells must be ...

Metabolic Labeling of Leucine Rich Repeat Kinases 1 and 2 with Radioactive Phosphate

Metabolic Labeling of Leucine Rich Repeat Kinases 1 and 2 with Radioactive Phosphate (Video) | JoVE

Leucine rich repeat kinases 1 and 2 (LRRK1 and LRRK2) are paralogs which share a similar domain organization, including a serine-threonine kinase domain, ...

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures (Video) | JoVE

Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling ...

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

Temporal Tracking of Cell Cycle Progression Using Flow Cytometry without the Need for Synchronization (Video) | JoVE

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

LED Thermo Flow &#8212; Combining Optogenetics with Flow Cytometry

LED Thermo Flow Ã¢â‚¬â€ Combining Optogenetics with Flow Cytometry (Video) | JoVE

Optogenetic tools allow isolated, functional investigations of almost any signaling molecule within complex signaling pathways. A major obstacle is the ...