Name: generation and isolation of cell cycle-arrested cells with complex karyotypes
Uploaded: 2018-04-13T13:00:00.000+00:00
Duration: 5 min 22 s
Description: Watch this Scientific Journal Video about Generation and Isolation of Cell Cycle-arrested Cells with Complex Karyotypes at JoVE.com

This work was supported in part by the Koch Institute Support (core) Grant P30-CA 14051 from the National Cancer Institute, by the National Institutes of Health Grant (CA206157-22 and GM118066) and Curt Marble Cancer Research Fund to Angelika Amon. Angelika Amon is also an investigator of the Howard Hughes Medical Institute and the Glenn Foundation for Biomedical Research. S. S. was supported by the American Italian Cancer Foundation (AICF), by a Fellowship in Cancer Research from Marie Curie Actions and the Italian Association for Cancer Research (AIRC), and by a KI Quinquennial Cancer Research Fellowship. E.M. was supported by a fellowship from the Howard Hughes Medical Institute.

Culture condition
RPE-1 hTERT cell line was cultured in Dulbecco&#39;s Modified Eagle Medium (Table of Materials) supplemented with 10% Fetal Bovine Serum, 2 mM L-glutamine, and 100 U/ml penicillin/streptomycin. Cells were incubated at 37 &#176;C with 5% CO2 in a humidified environment. The protocol described below has been developed by using 10-cm dishes and all technical details reported refer to those dishes. If using different dishes, scale up or down accordingly.
1. Synchronization of RPE-1 cells
<ol>
	<li>Use freshly thawed cells that have not undergone more than 15 passages for all experiments. On day 1, thaw and dispense ~2.5 - 5.0 x 105 RPE-1 hTERT cells into a 10-cm tissue culture dish. Add 8 mL of standard growth medium and incubate overnight. Determine cell number using a standard cell counter (such as a hemocytometer). 
	NOTE: Cell density refers to growth conditions of RPE-1 cells. Growth conditions might vary among different cell lines; consider adjusting them accordingly.</li>
	<li>On day 2, synchronize cells at the G1/S border by aspirating regular growth medium and adding 8 mL medium containing 5 mM thymidine. 
	NOTE: Thymidine concentration and length of treatment might vary across different cell lines. It is advisable to perform pilot experiments to determine the best conditions for the cell line(s) to be tested.</li>
	<li>On day 3, 24 h after adding thymidine medium (step 1.2), release from thymidine block by aspirating medium and washing cells three times with 1x PBS. Add 8 mL of regular growth medium and return plates to the incubator.</li>
</ol>
2. Generation of Aneuploid Cells by Interference with the Activity of the Mitotic Kinase Mps1
<ol>
	<li>On day 3, 6 h after release from thymidine block (step 1.3; this is about 3 - 6 h before cells enter mitosis), aspirate medium, wash once with 1x PBS, and replace with medium containing 500 nM reversine (the Mps1 inhibitor). 
	NOTE: RPE-1 cells enter mitosis 9 - 12 h after thymidine wash-out10,11. However, cell cycle kinetics vary among different cell lines. Therefore, it is crucial to determine those kinetics in pilot experiments by performing cell cycle analysis by established methods (e.g., by measuring DNA content via FACS analysis). Additionally, the optimal working concentration of Mps1 inhibitor may vary between cell lines. Previous experiments using RPE1 cells have been successful using reversine at a working concentration of 500 nM10,11,12. It is advisable to perform pilot experiments to determine the optimal concentration for the cell line to be tested.</li>
	<li>On day 4, 12 h after reversine treatment (about 6 h after cells were in mitosis), aspirate reversine medium and wash cells three times with 1x PBS. Add 8 mL regular growth medium to the plate and return cells to the incubator.</li>
</ol>
3. Removal of Aneuploid Cycling Cells and Enrichment of ArCK Population
<ol>
	<li>On day 6, about 72 h after cells enter the first mitosis (step 2.2; this is about 66 h after reversine wash-out, which corresponds to about 2 - 3 cell cycles), aspirate medium, wash once with 1x PBS and replace with medium containing 300 nM nocodazole. 
	NOTE: Recent work has shown that RPE-1 cells harboring aneuploid karyotypes are genomically unstable and arrest their proliferation about 2 - 3 cell cycles after the first faulty mitosis10. However, this kinetics might vary among different cell lines. Therefore, it is crucial to perform pilot experiments to determine the proper timing for the removal of aneuploid cycling cells.</li>
	<li>On day 7, 12 h after nocodazole treatment, aspirate medium and add 3 mL of 1x PBS to the plate. Shake off the mitotic cells by tapping the side of the plate. Rotate the plate between each tap to allow even removal of mitotic cells.</li>
	<li>To ensure complete removal of mitotic cells, repeat the shake-off process for multiple rounds (3 - 5 times recommended), aspirating and adding 3 mL of 1x PBS between each round.</li>
	<li>After shake-off, gently aspirate any liquid that adhered to the lid or the edge of the plate. Briefly, check the plate under a microscope at 10X magnification. Ensure that few mitotic cells are observed. If more than five mitotic cells are seen under 10X objective, repeat another round of shake-off. 
	NOTE: Mitotic cells would appear rounded and may be partially detached after shake-off. It is crucial to ensure the complete removal of mitotic cells before exposing the cells to the next round of nocodazole treatment. Failure to do so may lead to mitotic cell death and/or result in the generation of tetraploid cells.</li>
	<li>After final shake-off, wash cells once with 5 mL of 1x PBS. Add 8 mL medium containing 330 nM nocodazole into the plate and incubate for 12 h.</li>
	<li>Repeat nocodazole treatment/shake-off (steps 3.1 - 3.5) every 12 h until no mitotic cells are observed after incubation. Usually, 4-5 rounds of treatment/shake-off are recommended. 
	NOTE: The information provided here refer to RPE-1 cells. The number of rounds of treatment/shake-off might vary among different cell lines. To avoid prolonged and unnecessary exposure to nocodazole it is crucial to carefully determine, in a pilot experiment, the number of rounds required for efficient and complete removal of aneuploid cycling cells.</li>
	<li>After 4 - 5 rounds of nocodazole treatment/shake-off (steps 3.1 - 3.6), add 10 mL of regular growth medium and incubate. Allow cells to rest for 12 - 24 h before future manipulation or assay use. 
	NOTE: After final shake-off, cells remaining on the plate are cell cycle arrested and, as shown recently10, harbor complex karyotypes. These cells are referred to as ArCK (Arrested with Complex Karyotype) cells. It is advisable to perform assays on ArCK populations within a week of isolation.</li>
	<li>Optionally, to assess the percentage of arrested cells with complex karyotypes, collect and count cells from each shake-off using a standard cell counter.</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57215/57215fig1.jpg" /> 
Figure 1: Overall schematic of the method used to generate and isolate aneuploid cells with complex karyotype (ArCK) and representative images. (A) Schematic of ArCK cells generation and isolation protocol. (B) Representative images of RPE-1 hTERT cells at each stage of the treatment. The last panel (outlined in red) represents the isolated ArCK cells. Scale bar 100 &#181;m. <a href="//cloudfront.jove.com/files/ftp_upload/57215/57215fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
4. Characterization of ArCK Population
<ol>
	<li>Confirm successful isolation of ArCK population by performing the following assays.
	<ol>
		<li>Asses beta-galactosidase staining (the marker of senescence) using a commercially available kit (see Table of Materials).</li>
		<li>Measure secretion of pro-inflammatory cytokines, such as CCL2, by employing commercially available kits (see Table of Materials).</li>
		<li>Confirm increased levels of the cell cycle inhibitors p53, p21, p16 by Western Blot. 
		NOTE: To properly control these assays, ArCK populations should be compared to both euploid and aneuploid cycling cells. The former is low passage, wild-type RPE-1 hTERT cells exponentially growing. The latter can be obtained by inducing chromosome mis-segregation in RPE-1 hTERT and harvesting cells 72 h after the first aberrant mitosis. To do so, follow the protocol described above from step 1.1 and harvest cells right before the first nocodazole treatment (step 3.1).</li>
	</ol>
	</li>
	<li>Perform single-cell sequencing to assess the karyotype of the arrested cells. 
	NOTE: Single-cell sequencing is a well-established method to determine chromosomal and sub-chromosomal changes across a cell population at the single cell level13. Detailed procedures for how to perform single-cell sequencing can be found elsewhere10.</li>
</ol>

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	<li>Santaguida, S., Amon, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Short-+and+long-term+effects+of+chromosome+mis-segregation+and+aneuploidy.">Short- and long-term effects of chromosome mis-segregation and aneuploidy.</a> Nature Reviews Molecular Cell Biology. 16 (8), 473-485 (2015).</li><li>Pfau, S. J., Amon, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromosomal+instability+and+aneuploidy+in+cancer:+from+yeast+to+man.">Chromosomal instability and aneuploidy in cancer: from yeast to man.</a> EMBO reports. 13 (6), 515-527 (2012).</li><li>Gordon, D. J., Resio, B., Pellman, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Causes+and+consequences+of+aneuploidy+in+cancer.">Causes and consequences of aneuploidy in cancer.</a> Nature Reviews Genetics. 13 (3), 189-203 (2012).</li><li>Holland, A. J., Cleveland, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Boveri+revisited:+chromosomal+instability,+aneuploidy+and+tumorigenesis.">Boveri revisited: chromosomal instability, aneuploidy and tumorigenesis.</a> Nature Reviews Molecular Cell Biology. 10 (7), 478-487 (2009).</li><li>Meena, J. K., Cerutti, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Telomerase+abrogates+aneuploidy-induced+telomere+replication+stress,+senescence+and+cell+depletion.">Telomerase abrogates aneuploidy-induced telomere replication stress, senescence and cell depletion.</a> The EMBO Journal. , 1-14 (2015).</li><li>Ohashi, A., Ohori, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aneuploidy+generates+proteotoxic+stress+and+DNA+damage+concurrently+with+p53-mediated+post-mitotic+apoptosis+in+SAC-impaired+cells.">Aneuploidy generates proteotoxic stress and DNA damage concurrently with p53-mediated post-mitotic apoptosis in SAC-impaired cells.</a> Nature Communications. 6, 7668 (2015).</li><li>Passerini, V., Ozeri-Galai, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+presence+of+extra+chromosomes+leads+to+genomic+instability.">The presence of extra chromosomes leads to genomic instability.</a> Nature Communications. 7, 10754 (2016).</li><li>Sheltzer, J. M., Blank, H. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aneuploidy+drives+genomic+instability+in+yeast.">Aneuploidy drives genomic instability in yeast.</a> Science. 333 (6045), 1026-1030 (2011).</li><li>Zhu, J., Pavelka, N., Bradford, W. D., Rancati, G., Li, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Karyotypic+determinants+of+chromosome+instability+in+aneuploid+budding+yeast.">Karyotypic determinants of chromosome instability in aneuploid budding yeast.</a> PLoS Genetics. 8 (5), e1002719 (2012).</li><li>Santaguida, S., Richardson, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromosome+Mis-segregation+Generates+Cell-Cycle-Arrested+Cells+with+Complex+Karyotypes+that+Are+Eliminated+by+the+Immune+System.">Chromosome Mis-segregation Generates Cell-Cycle-Arrested Cells with Complex Karyotypes that Are Eliminated by the Immune System.</a> Developmental Cell. 41 (6), 638.e5-651.e5 (2017).</li><li>Santaguida, S., Vasile, E., White, E., Amon, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aneuploidy-induced+cellular+stresses+limit+autophagic+degradation.">Aneuploidy-induced cellular stresses limit autophagic degradation.</a> Genes &amp; Development. 29 (19), 2010-2021 (2015).</li><li>Santaguida, S., Tighe, A., D'Alise, A. 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The authors have nothing to disclose.

This novel method to generate and enrich for arrested cells with complex karyotypes (ArCK) allows for the study of cells that have multiple chromosome gains or losses and cease to divide. The method setup has been designed to facilitate the isolation of ArCK cells in a quick and reliable way.
The most critical step in this assay is rigorously controlling the timeline of drug treatment and, most importantly, the removal of aneuploid cycling cells. For optimal results, the timing of nocodazole treatment and shake-off is of particular importance to ensure that cycling cells are removed from the plate while they are still rounded and mitotic, preventing the possibility of mitotic cell death or slippage into G1, potentially creating a tetraploid population. It is not recommended that the timing of shake-off deviates more than two hours from the recommended 12-h nocodazole incubation.
Future studies on ArCK cells have the potential to facilitate a deeper understanding of how complex karyotypes affect cell physiology. In particular, a recent study demonstrated that cells of the immune system are able to interact with and trigger immune clearance of ArCK cells10. The method described here provides an excellent starting point for further characterization of ArCK cells, including the clarification of the molecular mechanisms underlying immune clearance in untransformed cells and the study of how oncogenic transformation may bypass this surveillance mechanism, a crucial question in the field14,15.

Errors in the process of chromosome segregation lead to aneuploidy, a condition characterized by a chromosome number that is not a multiple of the haploid complement1,2,3,4. Aneuploid karyotypes trigger replication stress that generates further genomic instability5,6,7,8,9,10, increases karyotype complexity, and ultimately leads to cell cycle arrest of a subpopulation of cells10. The purpose of the method presented here is to generate and separate such a subpopulation from cycling cells. By employing inexpensive tissue culture techniques, this protocol facilitates the isolation and characterization of cell cycle-arrested aneuploid cells with complex karyotypes. These cells are referred to as ArCK (Arrested with Complex Karyotype) cells and their euploid cycling counterparts as controls.
This protocol is the first one to be established for this purpose and allows for the isolation and further study of ArCK lines including, but not limited to, their induced senescence and the senescence-associated secretory phenotype (SASP), their pro-inflammatory features, and their ability to engage with immune cells. The method presented here has been developed in untransformed, immortalized human cells but has not yet been tested in cancer lines. Some transformed cells may be insensitive to cell cycle arrest due to suppression of one or multiple pathways; therefore, further validation should be performed in other cell lines.

<table><tbody><tr><td>DMEM (Dulbecco&amp;rsquo;s Modified Eagle Medium)&amp;nbsp;</td><td>Thermofisher</td><td>11995-073</td><td></td></tr><tr><td>Thymidine</td><td>Sigma Aldrich</td><td>T1859</td><td></td></tr><tr><td>Reversine</td><td>Cayman Chemical Company</td><td>10004412</td><td></td></tr><tr><td>Nocodazole</td><td>Sigma Aldrich</td><td>M1410</td><td></td></tr><tr><td>Anti-p53 antibody</td><td>Santa Cruz</td><td>sc-126</td><td></td></tr><tr><td>Anti-p21 antibody</td><td>Santa Cruz</td><td>sc-6246</td><td></td></tr><tr><td>Anti-p16 antibody</td><td>BD</td><td>554079</td><td></td></tr><tr><td>Senescence b-Galactosidase Staining Kit</td><td>Cell Signaling Technology&amp;nbsp;</td><td>9860</td><td></td></tr><tr><td>Proteome Profiler Human Cytokine Array Kit&amp;nbsp;</td><td>R&amp;amp;D Systems</td><td>ARY005B</td><td></td></tr></tbody></table>

generation and isolation of cell cycle-arrested cells with complex karyotypes

Chromosome mis-segregation leads to aneuploidy, a condition in which cells harbor an imbalanced chromosome number. Several lines of evidence strongly indicate that aneuploidy triggers genome instability, ultimately generating cells with complex karyotypes that arrest their proliferation. Isolation and characterization of cells harboring complex karyotypes are crucial to study the impact of an imbalanced chromosome number on cell physiology. To date, no methods have been established to reliably isolate such aneuploid cells. This paper provides a protocol for the enrichment and analysis of aneuploid cells with complex karyotypes utilizing standard, inexpensive tissue culture techniques. This protocol can be used to analyze several features of aneuploid cells with complex karyotypes including their induced senescence-associated secretory phenotype, pro-inflammatory properties, and ability to interact with immune cells. Because cancer cells often harbor imbalances in chromosome number, it is crucial to decipher how aneuploidy impacts cell physiology in normal cells, with the ultimate goal of uncovering both its pro- and anti-tumorigenic effects.

This method utilizes an in vitro tissue culture system to isolate aneuploid cells with complex karyotypes that arrest their proliferation. This population is referred to as ArCK (Arrested with Complex Karyotypes) cells. Figure 1A shows the scheme of the experiment. Wild-type cycling RPE-1 hTERT cells are synchronized at the G1/S border with thymidine and treated with a Mps1 inhibitor to induce chromosome mis-segregation. The spindle poison nocodazole is added to the cells 72 h after induction of chromosome mis-segregation. 12 h after nocodazole treatment, the aneuploid cells that are still able to proliferate enter mitosis and are trapped in this cell cycle stage due to interference with microtubule polymerization. These trapped cells are then easily removed by gentle shake-off. The shake-off process is repeated 3 - 5 times to ensure complete removal of cycling cells. Figure 1B shows the representative images of RPE-1 cells at each stage of the treatment.
ArCK cells display increased levels of cell cycle inhibitors, such as p53, p21, and p16 compared to euploid and aneuploid cycling cells, as shown in Figure 2A. Additionally, Figure 2B shows positive &#946;-galactosidase staining in ArCK cells in comparison to euploid control cells, a well-established marker for cellular senescence.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57215/57215fig2.jpg" /> 
Figure 2: Representative results utilizing ArCK cells. (A) Western blot assessing cell cycle inhibitor levels in euploid, aneuploid cycling, and ArCK cells. The levels of p53, p21, and p16 were determined by western blot analysis. Actin served as a loading control. (B) &#946;-galactosidase staining assessing the level of senescence in ArCK cells. Senescence-associated &#946;-galactosidase (&#946;-Gal) activity was determined in euploid cells and ArCK cells. Scale bar 100 &#181;m. <a href="https://cloudflare.jove.com/files/ftp_upload/57215/57215fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
This cell cycle arrest is a prominent feature of aneuploid cells with complex karyotypes, as shown by representative karyotype analysis from single-cell sequencing in Figure 3, in which ArCK cells display multiple, random chromosome gains and losses. This finding is in full agreement with previous reports10.
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57215/57215fig3.jpg" /> 
Figure 3: Representative single-cell sequencing of ArCK cells. Segmentation plots showing the karyotype of six representative ArCK cells. Segmentation plots show the copy number of all chromosomes from 1 to X relative to a euploid reference on a log2 scale (wild type RPE-1 is a diploid cell line of female origin). Chromosome gains are highlighted in red, chromosome losses in green. <a href="https://cloudflare.jove.com/files/ftp_upload/57215/57215fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Generation and Isolation of Cell Cycle-arrested Cells with Complex Karyotypes at JoVE.com

Generation and Isolation of Cell Cycle-arrested Cells with Complex Karyotypes

Aneuploidy leads to genome instability, which eventually produces cell cycle-arrested cells with complex karyotypes. This paper provides a simple and convenient method to isolate aneuploid cells with complex karyotypes that cease to divide.

Chromosome mis-segregation leads to aneuploidy, a condition in which cells harbor an imbalanced chromosome number. Several lines of evidence strongly ...

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Cancer Research

10.4K Views.  Massachusetts Institute of Technology. Aneuploidy leads to genome instability, which eventually produces cell cycle-arrested cells with complex karyotypes. This paper provides a simple and convenient method to isolate aneuploid cells with complex karyotypes that cease to divide.

Video: Generation and Isolation of Cell Cycle-arrested Cells with Complex Karyotypes

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.

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

Genetics

Preparation of Primary Acute Lymphoblastic Leukemia Cells in Different Cell Cycle Phases by Centrifugal Elutriation

The ability to synchronize cells has been central to advancing our understanding of cell cycle regulation. Common techniques employed include serum deprivation; chemicals which arrest cells at different cell cycle phases; or the use of mitotic shake-off which exploits their reduced adherence. However, all of these have disadvantages. For example, serum starvation works well for normal cells but less well for tumor cells with compromised cell cycle checkpoints due to oncogene activation or tumor suppressor loss. Similarly, chemically-treated cell populations can harbor drug-induced damage and show stress-related alterations. A technique which circumvents these problems is counterflow centrifugal elutriation (CCE), where cells are subjected to two opposing forces, centrifugal force and fluid velocity, which results in the separation of cells on the basis of size and density. Since cells advancing through the cycle typically enlarge, CCE can be used to separate cells into different cell cycle phases. Here we apply this technique to primary acute lymphoblastic leukemia cells. Under optimal conditions, an essentially pure population of cells in G1 phase and a highly enriched population of cells in G2/M phases can be obtained in excellent yield. These cell populations are ideally suited for studying cell cycle-dependent mechanisms of action of anticancer drugs and for other applications. We also show how modifications to the standard procedure can result in suboptimal performance and discuss the limitations of the technique. The detailed methodology presented should facilitate application and exploration of the technique to other types of cells.

This protocol describes the use of centrifugal elutriation to separate primary acute lymphoblastic leukemia cells into different cell cycle phases.

The ability to synchronize cells has been central to advancing our understanding of cell cycle regulation. Common techniques employed include serum ...

Studying RNA Interactors of Protein Kinase RNA-Activated during the Mammalian Cell Cycle

Protein kinase RNA-activated (PKR) is a member of the innate immune response proteins and recognizes the double-stranded secondary structure of viral RNAs. When bound to viral double-stranded RNAs (dsRNAs), PKR undergoes dimerization and subsequent autophosphorylation. Phosphorylated PKR (pPKR) becomes active and induces phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF2&#945;) to suppress global translation. Increasing evidence suggests that PKR can be activated under physiological conditions such as during the cell cycle or under various stress conditions without infection. However, our understanding of the RNA activators of PKR is limited due to the lack of a standardized experimental method to capture and analyze PKR-interacting dsRNAs. Here, we present an experimental protocol to specifically enrich and analyze PKR bound RNAs during the cell cycle using HeLa cells. We utilize the efficient crosslinking activity of formaldehyde to fix PKR-RNA complexes and isolate them via immunoprecipitation. PKR co-immunoprecipitated RNAs can then be further processed to generate a high-throughput sequencing library. One major class of PKR-interacting cellular dsRNAs is mitochondrial RNAs (mtRNAs), which can exist as intermolecular dsRNAs through complementary interaction between the heavy-strand and the light-strand RNAs. To study the strandedness of these duplex mtRNAs, we also present a protocol for strand-specific qRT-PCR. Our protocol is optimized for the analysis of PKR-bound RNAs, but it can be easily modified to study cellular dsRNAs or RNA-interactors of other dsRNA binding proteins.

We present experimental approaches for studying RNA-interactors of double-stranded RNA binding protein kinase RNA-activated (PKR) during the mammalian cell cycle using HeLa cells. This method utilizes formaldehyde to crosslink RNA-PKR complexes and immunoprecipitation to enrich PKR-bound RNAs. These RNAs can be further analyzed through high-throughput sequencing or qRT-PCR.

Protein kinase RNA-activated (PKR) is a member of the innate immune response proteins and recognizes the double-stranded secondary structure of viral ...

Biochemistry

Live Cell Imaging of Chromosome Segregation During Mitosis

Chromosomes must be reliably and uniformly segregated into daughter cells during mitotic cell division. Fidelity of chromosomal segregation is controlled by multiple mechanisms that include the Spindle Assembly Checkpoint (SAC). The SAC is part of a complex feedback system that is responsible for prevention of a cell progress through mitosis unless all chromosomal kinetochores have attached to spindle microtubules. Chromosomal lagging and abnormal chromosome segregation is an indicator of dysfunctional cell cycle control checkpoints and can be used to measure the genomic stability of dividing cells. Deregulation of the SAC can result in the transformation of a normal cell into a malignant cell through the accumulation of errors during chromosomal segregation. Implementation of the SAC and the formation of the kinetochore complex are tightly regulated by interactions between kinases and phosphatase such as Protein Phosphatase 2A (PP2A). This protocol describes live cell imaging of lagging chromosomes in mouse embryonic fibroblasts isolated from mice that had a knockout of the PP2A-B56&#947; regulatory subunit. This method overcomes the shortcomings of other cell cycle control imaging techniques such as flow cytometry or immunocytochemistry that only provide a snapshot of a cell cytokinesis status, instead of a dynamic spatiotemporal visualization of chromosomes during mitosis.

This protocol describes an easy and convenient method to label and visualize live chromosomes in mitotic cells using Histone2B-GFP BacMam 2.0 label and a spinning disc confocal microscopy system.

Chromosomes must be reliably and uniformly segregated into daughter cells during mitotic cell division. Fidelity of chromosomal segregation is controlled ...

Developmental Biology

Chromosomal Spread Preparation of Human Embryonic Stem Cells for Karyotyping

Although human embryonic stem cells (hESC) have been shown to present a stable diploid karyotype 1, many studies have reported that depending on culture conditions they become prone to acquire chromosomal anomalies such as addition of whole or parts of chromosomes. Indeed, during long-term culture, karyotypic alterations are observed when enzymatic or chemical dissociation are used 2,3,4, while manual dissection of colonies for passaging retains a stable karyotype 5. Besides, changes in the environment such as the removal of feeder cells also seem to compromise the genetic integrity of hESC 3,6. Once chromosomal alterations could affect cellular physiology, the characterization of the genetic integrity of hESC in vitro is crucial considering hESC as an essential tool in embryogenesis studies and drug testing. Furthermore, for future therapeutic purposes chromosomal changes are a real concern as it is frequently associated to carcinogenesis.
Here we show a simple and useful method to obtain high quality chromosome spreads for subsequent analysis of chromosome set by G-banding, FISH, SKY or CGH techniques 7,8. We recommend checking the chromosomal status routinely with intervals of 5 passages in order to monitor the appearance of translocations and aneuploidies
Priscila Britto and Rafaela Sartore contributed equally to the paper.

Karyotyping is a simple and useful technique widely used for detecting genetic alterations. Here we describe a step by step protocol for chromosome spread preparation of human embryonic stem cells for monitoring the chromosomal status of these cells maintained in culture.

Although human embryonic stem cells (hESC) have been shown to present a stable diploid karyotype 1, many studies have reported that depending on culture ...

Biology

Adult and Embryonic Skeletal Muscle Microexplant Culture and Isolation of Skeletal Muscle Stem Cells

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a robust and reliable method for isolating relatively large numbers of proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. The authors have used micro-dissected explant cultures to analyse the growth characteristics of skeletal muscle cells in wild-type and dystrophic muscles. Each of the components of tissue growth, namely cell survival, proliferation, senescence and differentiation can be analysed separately using the methods described here. The net effect of all components of growth can be established by means of measuring explant outgrowth rates. The micro-explant method can be used to establish primary cultures from a wide range of different muscle types and ages and, as described here, has been adapted by the authors to enable the isolation of embryonic skeletal muscle precursors.
Uniquely, micro-explant cultures have been used to derive clonal (single cell origin) skeletal muscle stem cell (SMSc) lines which can be expanded and used for in vivo transplantation. In vivo transplanted SMSc behave as functional, tissue-specific, satellite cells which contribute to skeletal muscle fibre regeneration but which are also retained (in the satellite cell niche) as a small pool of undifferentiated stem cells which can be re-isolated into culture using the micro-explant method.

The micro-dissected explants technique is a robust and reliable method for isolating proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. Uniquely, these cells have been clonally derived to produce skeletal muscle stem cell lines used for in vivo transplantation.

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a ...

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.

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of ...

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.

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.

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

Chromosome Preparation From Cultured Cells

Chromosome (cytogenetic) analysis is widely used for the detection of chromosome instability. When followed by G-banding and molecular techniques such as fluorescence in situ hybridization (FISH), this assay has the powerful ability to analyze individual cells for aberrations that involve gains or losses of portions of the genome&#160;and rearrangements involving one or more chromosomes. In humans, chromosome abnormalities occur in approximately 1 per 160 live births1,2, 60-80% of all miscarriages3,4, 10% of stillbirths2,5, 13% of individuals with congenital heart disease6, 3-6% of infertility cases2, and in many patients with developmental delay&#160;and birth defects7. Cytogenetic analysis of malignancy is routinely used by researchers and clinicians, as observations of clonal chromosomal abnormalities have been shown to have both diagnostic and prognostic significance8,9.&#160; Chromosome isolation is invaluable for gene therapy and stem cell research of organisms including nonhuman primates and rodents10-13.
Chromosomes can be isolated from cells of live tissues, including blood lymphocytes, skin fibroblasts, amniocytes, placenta, bone marrow, and tumor specimens. Chromosomes are analyzed at the metaphase stage of mitosis, when they are most condensed and therefore more clearly visible. The first step of the chromosome isolation technique involves the disruption of the spindle fibers by incubation with Colcemid, to prevent the cells from proceeding to the subsequent anaphase stage. The cells are then treated with a hypotonic solution and preserved in their swollen state with Carnoy&#39;s fixative. The cells are then dropped on to slides and can then be utilized for a variety of procedures. G-banding involves trypsin treatment followed by staining with Giemsa to create characteristic light and dark bands. The same procedure to isolate chromosomes can be used for the preparation of cells for procedures such as fluorescence in situ hybridization (FISH), comparative genomic hybridization (CGH), and spectral karyotyping (SKY)14,15.

Chromosomes can be isolated from live cells such as lymphocytes or skin fibroblasts, and from organisms including humans or mice. These chromosome preparations can be further utilized for routine G-banding and molecular cytogenetic procedures such as fluorescence in situ hybridization (FISH), comparative genomic hybridization (CGH), and spectral karyotyping (SKY).

Chromosome (cytogenetic) analysis is widely used for the detection of chromosome instability. When followed by G-banding and molecular techniques such as ...

A Cell Free Assay to Study Chromatin Decondensation at the End of Mitosis

During the vertebrate cell cycle chromatin undergoes extensive structural and functional changes. Upon mitotic entry, it massively condenses into rod shaped chromosomes which are moved individually by the mitotic spindle apparatus. Mitotic chromatin condensation yields chromosomes compacted fifty-fold denser as in interphase. During exit from mitosis, chromosomes have to re-establish their functional interphase state, which is enclosed by a nuclear envelope and is competent for replication and transcription. The decondensation process is morphologically well described, but in molecular terms poorly understood: We lack knowledge about the underlying molecular events and to a large extent the factors involved as well as their regulation. We describe here a cell-free system that faithfully recapitulates chromatin decondensation in vitro, based on mitotic chromatin clusters purified from synchronized HeLa cells and X. laevis egg extract. Our cell-free system provides an important tool for further molecular characterization of chromatin decondensation and its co-ordination with processes simultaneously occurring during mitotic exit such as nuclear envelope and pore complex re-assembly.

The molecular mechanisms of the decondensation of highly compacted mitotic chromatin are ill-defined. We present a cell-free assay based on mitotic chromatin clusters isolated from HeLa cells and Xenopus laevis egg extract that faithfully reconstitutes the decondensation process in vitro.

During the vertebrate cell cycle chromatin undergoes extensive structural and functional changes. Upon mitotic entry, it massively condenses into rod ...