The work was funded by the Leukemia and Lymphoma Society of the USA (6105-08), a Cancer Council NSW grant (13-02), an NHMRC Senior Research Fellowship (LJB) (1042305) and project grant (1041614).

The protocol described here uses the acute lymphoblastic leukemia cell line NALM6 but can be applied to other cell types.
1. Solutions and Reagents
<ol>
	<li>Complete RPMI
	<ol>
		<li>Add 56 ml fetal calf serum (FCS) and 5.5 ml of 200 mM L-glutamine to a 500 ml bottle of RPMI-1640 medium.</li>
	</ol>
	</li>
	<li>BrdU Stock Solution
	<ol>
		<li>Prepare 32.5 mM BrdU (10 mg/ml) in Dulbecco&#39;s Phosphate Buffered Saline (DPBS).</li>
	</ol>
	</li>
	<li>BrdU Complete RPMI
	<ol>
		<li>Add 6.2 &#181;l of BrdU stock solution to 10 ml of Complete RPMI.</li>
	</ol>
	</li>
	<li>DNase Solution
	<ol>
		<li>Prepare 1 mg DNase/ml in DPBS.</li>
	</ol>
	</li>
	<li>Staining Buffer
	<ol>
		<li>Prepare 3% heat-inactivated FCS and 0.09% sodium azide in DPBS.</li>
	</ol>
	</li>
	<li>Refer to Materials List for definitions of Fixation Buffer, Permeabilization Buffer and Wash Buffer.</li>
</ol>
2. Cells
Note: Cells were not cultured for greater than 6 months. This method is directly adaptable to any non-adherent cell line with adjustments to cell density and culture media. Use cells that are growing exponentially at the initiation of the experiment.
<ol>
	<li>Maintain NALM6 cells in T-75 culture flasks in Complete RPMI. Perform all steps under sterile conditions using a Class II Biosafety Cabinet.
	<ol>
		<li>Maintain NALM6 cells between 1-2 x 106 cells per ml by splitting the culture thrice weekly.</li>
		<li>Incubate at 37 &#176;C in 5% CO2 in air.</li>
	</ol>
	</li>
</ol>
3. Pulse Labeling of Cells with BrdU
CAUTION: Handle BrdU with care as it is a potential mutagen and teratogen.
<ol>
	<li>Centrifuge cells at 150 x g for 5 min. Note: Transferring cells into fresh media improves the reproducibility of the results.</li>
	<li>Perform a cell count and resuspend cells in Complete RPMI at 2 x 106 cells/ml.</li>
	<li>Dilute cells 1 in 2 with BrdU Complete RPMI producing a final cell concentration of 1 x 106 cells/ml.</li>
	<li>Incubate at 37 &#176;C with 5% CO2 for 45 min, then dilute cells 1 in 10 with complete RPMI. Centrifuge cells at 150 x g for 5 min and carefully discard all of the supernatant.</li>
	<li>Resuspend cells in a small volume (~100 &#181;l) of complete RPMI, perform a cell count and adjust to 1 x 106 cells/ml.</li>
	<li>Pipette 1 ml of cells into the wells of a 48 well plate. Pipette 1 ml of DPBS into any unoccupied wells to obtain more reproducible results.</li>
	<li>Incubate at 37 &#176;C in 5% CO2 in air for desired timepoints, here 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, and 24 hr. Note: The length of time will depend on what the experimental design aims to measure.</li>
	<li>Transfer all the cells into FACS tubes using a pipette. Rinse the well sequentially with 1 ml volumes of PBS to a final total volume of 5 ml.</li>
	<li>Centrifuge at 150 x g for 5 min and carefully remove all the supernatant. Cells are ready for staining, perform this (section 4) immediately.</li>
</ol>
4. Cell Staining
Note: If surface staining of cells is required perform it prior to fixation, ensuring that the cells are kept at 4 &#176;C throughout.
<ol>
	<li>Resuspend cells in 100 &#181;l of staining buffer (for optional surface staining, add the recommended volume of antibody to surface antigens and incubate for 30 min at 4 &#176;C).</li>
	<li>Add 1 ml of staining buffer, centrifuge for 5 min at 150 x g and discard the supernatant. 
	Note: Specific antibody, concentration, incubation time etc. will vary depending on specific experimental goals.</li>
	<li>Fixation and Permeabilization
	<ol>
		<li>Resuspend cells in 100 &#181;l of fixation buffer and incubate for 15 min at room temperature.</li>
		<li>Add 1 ml of wash buffer, centrifuge for 5 min at 150 x g and discard the supernatant.</li>
		<li>Resuspend cells in 100 &#181;l of permeabilization buffer and incubate the cells for 10 min on ice.</li>
		<li>Add 1 ml of wash buffer, centrifuge for 5 min at 150 x g, and discard the supernatant.</li>
		<li>Resuspend cells in 100 &#181;l of fixation buffer per tube and incubate for 5 min at room temperature.</li>
		<li>Add 1 ml of wash buffer, centrifuge for 5 min at 150 x g, and discard the supernatant. 
		Note: The protocol can be paused here if required. The fixed cells are stable for several days at 4 &#176;C if resuspended in staining buffer. Remove the staining buffer following centrifugation before proceeding.</li>
	</ol>
	</li>
	<li>DNase Treatment
	<ol>
		<li>Resuspend cells in 100 &#181;l of DNase solution (30 &#181;g of DNase/106 cells) and incubate cells for 1 hr at 37 &#176;C.</li>
		<li>Add 1 ml of wash buffer, centrifuge at 150 x g for 5 min and discard supernatant.</li>
	</ol>
	</li>
	<li>Antibody Staining 
	Note: Staining for intracellular markers other than BrdU can be performed simultaneously with the BrdU staining.
	<ol>
		<li>IMPORTANT: Prepare compensation controls consisting of unstained cells and cells labeled with each single fluorochrome. Ideally, use the same antibodies for compensation controls as those used in the experimental tubes. However, if this is not feasible, substitute antibodies to highly expressed antigens conjugated to the same fluorochrome.</li>
		<li>Resuspend the cells in 50 &#181;l of wash buffer and add 1 &#181;l/106 cells of BrdU antibody. Note: Directly conjugated antibodies to other specific intracellular antigens can also be added. &#160; 
		NOTE: Antibodies to histone H3 phosphorylated on Ser10 can be used to discriminate between cells in G2 and M, histone H3 is phosphorylated on Ser10 during mitosis.10 Antibodies to cdc2 phosphorylated on Tyr15 can be used to detect cells that have committed to mitosis.11</li>
		<li>Incubate the cells for 20 min at room temperature.</li>
		<li>Add 1 ml of wash buffer, centrifuge cells at 150 x g for 5 min and discard supernatant.</li>
	</ol>
	</li>
</ol>
<ol start="6">
	<li>Stain DNA for Cell Cycle Analysis
	<ol>
		<li>Loosen pellet and add 20 &#181;l of the 7-AAD solution (0.25 &#181;g). Note: It is critical to use a constant amount of 7-AAD/cell.</li>
		<li>Resuspend the cells in 1 ml of Staining buffer.</li>
	</ol>
	</li>
</ol>
5. Collection of Flow Cytometry Data
Note: The machine required will depend on the number and nature of the fluorochromes used.
<ol>
	<li>Collect the following parameters: FSC-A, SSC-A, FSC-H (FSC-W can be used instead of FSC-H) and 7-AAD fluorescence on a linear scale. Collect the APC channel on a log scale. Collect any additional channels required for the assessment of surface or internal labels using a log scale.</li>
	<li>Perform compensation of overlapping signals in emission spectra observed between different fluorochromes before analyzing the samples. Note: Most flow cytometers will perform this automatically.</li>
	<li>Collect at least 10,000 events for each sample.</li>
</ol>
6. Analysis of Flow Cytometry Data
Note: FlowJo was used in this study for flow cytometry data analysis but other software packages can also be used. The gating strategy is illustrated in Figure 1.
<ol>
	<li>Identify the viable cell population using FSC-A and SSC-A parameters.</li>
	<li>Within this population exclude doublets and aggregates using FSC-A and FSC-H (FSC-W can also be used here).</li>
	<li>Within this population set a dot plot using 7-AAD on the x-axis and BrdU-APC on the y-axis.</li>
</ol>
<img alt="Figure 1" src="/files/ftp_upload/52840/52840fig1highres.jpg" width="700" /> 
Figure 1: Gating Strategy. Left panel: ungated cells are shown on a FCS-A vs. SSC-A dot plot. The viable cell population is identified by the gate shown. Center panel: cells gated from the left panel are shown on a FSC-A vs. FSC-H dot plot (FSC-W can be used instead of height). Doublets and aggregates are identified, and excluded by the gate shown. Right panel: cells gated from the doublet exclusion date in the center panel are shown on a 7-AAD vs. APC-A dot plot. The BrdU antibody is labeled with APC permitting the identification of cells that have incorporated BrdU during the pulse labeling. 7-AAD provides information on DNA content. The upper gate defines cells positive for BrdU and therefore in S phase at the time of the BrdU pulse, the lower left gate, cells in G0/1 and the lower right gate those in G2/M. <a href="https://www.jove.com/files/ftp_upload/52840/52840fig1highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="4">
	<li>Cell Cycle Analysis
	<ol>
		<li>Open the first data file and gate on the cells in the doublet exclusion gate.</li>
		<li>Analyze this population for cell cycle distribution (located under platforms in FloJo software) and use the Dean-Jett-Fox model.</li>
		<li>Obtain the positions of the G0/1 and G2/M peaks using create gates.</li>
		<li>Gate on the BrdU positive cells and subject this population to same cell cycle analysis.</li>
		<li>Provide the positions for the G0/1 and G2/M peaks by applying the same gates from create gates and setting constraints (using the created gates) for the positions of the G0/1 and G2/M peaks. This is illustrated in the first 2 panels of Figure 2. 
		Note: Other software may also be used to analyze the data and the instructions would vary accordingly.</li>
	</ol>
	</li>
</ol>
<img alt="Figure 2" src="/files/ftp_upload/52840/52840fig2highres.jpg" width="700" /> 
Figure 2: Cell Cycle Progression. The first panel (All Cells) is gated on the cell population defined by the doublet exclusion gate. This population was displayed in a histogram with 7-AAD on the X-axis. The peak of the G0/1 peak is indicated by the arrow below the axis. In subsequent panels BrdU positive cells have been gated on as shown in Figure 1. The value for the G0/1 position obtained when gating of the doublet exclusion gate is applied to the BrdU positive gated cells within FlowJo cell cycle software. Each subsequent panel was gated on the BrdU positive population as shown in Figure 1 and the position of the G0/1 peak based on the value obtained when analyzing the whole population as shown in the first two panels. Using the BrdU negative fraction to identify the location of the G0/1 population for the BrdU positive cells in the same sample controls for any slight differences in the intensity of the DNA stain between samples. The number shown on each panel represents the time since the BrdU pulse ended. The calculated cell cycle phases are shown in shaded green. <a href="https://www.jove.com/files/ftp_upload/52840/52840fig2highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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The authors have nothing to disclose.

The ability to analyze the cell cycle is important for the understanding of cancer biology and the mechanism of action of both drugs and genes that influence cell proliferation and growth. While there are a multitude of assays that reportedly measure cell proliferation, the majority only provide a measure that indicates the number cells present. These include assays that measure cell number by direct visualization and counting, metabolic activity or ATP concentration. The main advantage of many of these methods is that t...

The assessment of cell cycle features and changes that occur in cells during cell cycle progression is fundamental to understanding many aspects of biology, particularly cancer biology. Many agents in development for the treatment of malignancies have profound effects on cell cycle progression or induce cell death via cell cycle dependent-mechanisms. In order to study cell cycle dynamics or cells in a particular phase of the cell cycle, it is usual to synchronize cells. However synchronization methods can have detrimental effects on the cells being studied, potentially confounding the results obtained.1 Recently the use of fluorescently tagged proteins that are only present at particular phases of the cells cycle have permitted analysis of cell cycle progression in single cells over time2, however the cells to be studied need to be genetically manipulated to express these tagged proteins, limiting their use to systems where this can be readily achieved.
The cell cycle consists of two active phases: the synthesis (S) phase, where DNA is replicated and mitosis (M) where cell division takes place. These phases are separated by three gap phases, G0, G1 and G2. G0 or quiescence, is a resting phase where the cell has left the cycle, G1 is where the cells increase in size prior to DNA replication and G2 where cell growth continues between completion of DNA replication but before cell division. The progression through the cell cycle is controlled by a number of checkpoints. The G1 checkpoint is activated when environmental conditions are not supportive of DNA synthesis and prevents entry into S phase. The intra-S phase checkpoint or delay can be triggered by DNA damage that may result in stalled replication forks. During G2 the fidelity of the replicated DNA is confirmed and if damage is detected then the G2 checkpoint is activated permitting DNA repair prior to cell division. A final checkpoint during mitosis ensures that chromatids have been correctly aligned at the mitotic plate so that cell division can be successfully completed.3 Activation of these checkpoints is commonly used to synchronize cell populations. Cell cycle checkpoints can be activated by a number of factors but in cancer biology the most common is detection of DNA damage. The DNA damage response is initiated by the PI3-kinase-like kinases ataxia telangiectasia and Rad3 related (ATR) and ataxia telangiectasia mutated (ATM) that activate the downstream effector kinases Chk1 and Chk2, respectively.3 A range of events activates Chk1 including stalled replication forks, DNA crosslinks, and ultraviolet radiation damage while Chk2 is primarily activated by double-strand breaks.
The usual method for studying the effect of altered conditions on the length of the cell cycle is to synchronize the cells in a particular phase of the cell cycle.1 This can be achieved via several methods. Cells can be physically separated based on size, density, side scatter (granularity), and cell surface expression markers. More practically, cells may be synchronized by chemical means. Several agents such as thymidine, hydroxyurea and cytosine arabinoside can be used to inhibit DNA synthesis in the S phase of cell cycle resulting in an accumulation of cells in S phase which continue cycling after the agents are removed. Cells treated with nocodazole, which prevents the formation of the mitotic spindle, arrest with a G2- or M-phase DNA content. Elimination of serum from the culture medium results in the accumulation of cells at G0 phase. The re-addition of the nutrients within the culture serum re-starts the normal cycling of the cells. However, all of these synchronization methods interfere with normal cycling and growth of cells and can result in significant cell death.
Synchronization of acute lymphoblastic leukemia cells is particularly challenging and these cells are not amenable to genetic manipulation. The method described here permits the assessment of cell cycle dynamics and the study of cells in particular phases of the cell cycle without traditional synchronization or genetic modification. This method may also be useful for other cell types where genetic modification and traditional synchronization procedures are not readily achieved. The method is based on the long established use of bromodeoxyuridine (BrdU) incorporation, which has very little impact on the short-term growth and proliferation of cells.4 Established BrdU protocols take advantage of the incorporation of BrdU into newly synthesized DNA during S phase. This permanently marks cells as having been in S phase during BrdU exposure. This population can be identified at later time points by staining for BrdU incorporation and thereby act as a synchronized population that can be followed and assessed over time permitting the study of drug effects on cell cycle transit. BrdU needs to be exposed prior to antibody staining, usually achieved following DNase or acid treatment.6,7 Using flow cytometry to detect incorporated BrdU enables the inclusion of additional markers. The most important is the use of dyes to measure DNA content, enabling the assessment of cell cycle phase distribution of the cells that were in S phase at the start of the study.8 Furthermore additional surface or intracellular antigens can also be studied.9 These may relate to cell cycle events such as Ki67 or to seemingly unrelated cell features such as apoptosis markers like cleaved caspase-3. The potential applications are limited by the imagination of the investigator.

<table><tbody><tr><td>APC BrdU Flow Kit</td><td>BD Biosciences</td><td>552598</td><td>Contains BrdU antibody, 7-AAD and BD Cytofix/Cytoperm&lt;br/&gt; Buffer (referred to as Fixation Buffer)</td></tr><tr><td>BD Cytoperm Permeabilization Buffer Plus</td><td>BD Biosciences</td><td>561651</td><td>Referred to as Permeabilization buffer</td></tr><tr><td>BD Perm/Wash Buffer</td><td>BD Biosciences</td><td>554723</td><td>Referred to as Wash buffer</td></tr><tr><td>DNase</td><td>Sigma</td><td>D-4513</td><td></td></tr><tr><td>BD Falcon 12 x 75&amp;nbsp;mm FACS tubes</td><td>BD Biosciences</td><td>352008</td><td></td></tr><tr><td>BD Pharmingen Stain Buffer</td><td>BD Biosciences</td><td>554656</td><td></td></tr><tr><td>BD LSR FORTESSA flow cytometer</td><td>BD Biosciences</td><td>FORTESSA</td><td></td></tr><tr><td>Pipetman</td><td>Gilson</td><td>P2, P20, P100, P1000</td><td></td></tr><tr><td>RPMI 1,640 w/o L-Gln 500 ml</td><td>Lonza</td><td>12-167F</td><td></td></tr><tr><td>DPBS</td><td>Lonza</td><td>17-512F</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>FisherBiotec</td><td>FBS-7100113</td><td></td></tr><tr><td>L-Glutamine</td><td>Sigma</td><td>G7513-100ML</td><td></td></tr><tr><td>5-Bromo-2&amp;prime;-deoxyuridine</td><td>Sigma</td><td>B5002-1G</td><td></td></tr><tr><td>Falcon TC 150&amp;nbsp;cm&lt;sup&gt;2&lt;/sup&gt; vented Flasks</td><td>BD Biosciences</td><td>355001</td><td></td></tr><tr><td>Pipettes 25&amp;nbsp;ml</td><td>Greiner</td><td>760180</td><td></td></tr><tr><td>Aersol Pipettes 200&amp;nbsp;&amp;micro;l</td><td>Interpath</td><td>24700</td><td></td></tr><tr><td>Aersol Pipettes 1&amp;nbsp;ml</td><td>Interpath</td><td>24800</td><td></td></tr><tr><td>Centrifuge</td><td>Spintron</td><td>GT-175R</td><td></td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt; incubator</td><td>Binder</td><td>C 150</td><td></td></tr><tr><td>AF488 anti-Histone H3 Phospho (Ser10) Antibody</td><td>Cell Signalling</td><td>9708S</td><td></td></tr><tr><td>Phospho-Chk2 (Thr68) (C13C1) Rabbit mAb</td><td>Cell Signalling</td><td>2197S</td><td></td></tr><tr><td>Phospho-Chk1 (Ser345) (133D3) Rabbit mAb</td><td>Cell Signalling</td><td>2348S</td><td></td></tr><tr><td>NALM6&amp;nbsp;</td><td>DSMZ</td><td>ACC-128</td><td></td></tr></tbody></table>

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 methodology can be used to obtain a range of information. A few applications are outlined here.
Assessment of the duration of the cell cycle
To determine the time required for cells to transit through the cell cycle, cells are harvested at various time points following the BrdU pulse. The intervals between assessments can be adapted to the particular cells being analyzed. Hematopoietic cell lines were assessed every hour over a 24 hr period in the absence of an...

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

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

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.

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

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19.2K Views.  Westmead Millennium Institute for Medical Research and University of Sydney.  Dit protocol beschrijft het gebruik van broomdeoxyuridine (BrdU) om de tijdelijke opname volgen van cellen die op een bepaald tijdstip waren in de S-fase mogelijk. Toevoeging van DNA kleurstoffen en antilichamen etikettering bevordert gedetailleerde analyse van het lot van de S-fase cellen op latere tijdstippen. 

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

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

Cell Cycle-specific Measurement of &#947;H2AX and Apoptosis After Genotoxic Stress by Flow Cytometry

The presented method or slightly modified versions have been devised to study specific treatment responses and side effects of various anti-cancer treatments as used in clinical oncology. It enables a quantitative and longitudinal analysis of the DNA damage response after genotoxic stress, as induced by radiotherapy and a multitude of anti-cancer drugs. The method covers all stages of the DNA damage response, providing endpoints for induction and repair of DNA double-strand breaks (DSBs), cell cycle arrest and cell death by apoptosis in case of repair failure. Combining these measurements provides information about cell cycle-dependent treatment effects and thus allows an in-depth study of the interplay between cellular proliferation and coping mechanisms against DNA damage. As the effect of many cancer therapeutics including chemotherapeutic agents and ionizing radiation is limited to or strongly varies according to specific cell cycle phases, correlative analyses rely on a robust and feasible method to assess the treatment effects on the DNA in a cell cycle-specific manner. This is not possible with single-endpoint assays and an important advantage of the presented method. The method is not restricted to any particular cell line and has been thoroughly tested in a multitude of tumor and normal tissue cell lines. It can be widely applied as a comprehensive genotoxicity assay in many fields of oncology besides radio-oncology, including environmental risk factor assessment, drug screening and evaluation of genetic instability in tumor cells.

The presented method combines the quantitative analysis of DNA double-strand breaks (DSBs), cell cycle distribution and apoptosis to enable cell cycle-specific evaluation of DSB induction and repair as well as the consequences of repair failure.

The presented method or slightly modified versions have been devised to study specific treatment responses and side effects of various anti-cancer ...

Cancer Research

Simultaneous Imaging and Flow-Cytometry-based Detection of Multiple Fluorescent Senescence Markers in Therapy-Induced Senescent Cancer Cells

Chemotherapeutic drugs can induce irreparable DNA damage in cancer cells, leading to apoptosis or premature senescence. Unlike apoptotic cell death, senescence is a fundamentally different machinery restraining&#160;propagation of cancer cells. Decades of scientific studies have revealed the complex pathological effects of senescent cancer cells in tumors and microenvironments that modulate cancer cells and stromal cells. New evidence suggests that senescence is a potent prognostic factor during cancer treatment, and therefore rapid and accurate detection of senescent cells in cancer samples is essential. This paper presents a method to visualize and detect therapy-induced senescence (TIS) in cancer cells. Diffuse large B-cell lymphoma (DLBCL) cell lines were treated with mafosfamide (MAF) or daunorubicin (DN) and examined for the senescence marker, senescence-associated &#946;-galactosidase (SA-&#946;-gal), the DNA synthesis marker 5-ethynyl-2&#8242;-deoxyuridine (EdU), and the DNA damage marker gamma-H2AX (&#947;H2AX). Flow cytometer imaging can help generate high-resolution single-cell images in a short period of time to simultaneously visualize and quantify the three markers in cancer cells.

Here we present a flow cytometry-based method for visualization and quantification of multiple senescence-associated markers in single cells.

Chemotherapeutic drugs can induce irreparable DNA damage in cancer cells, leading to apoptosis or premature senescence. Unlike apoptotic cell death, ...

Monitoring Kinase and Phosphatase Activities Through the Cell Cycle by Ratiometric FRET

F&ouml;rster resonance energy transfer (FRET)-based reporters1 allow the assessment of endogenous kinase and phosphatase activities in living cells. Such probes typically consist of variants of CFP and YFP, intervened by a phosphorylatable sequence and a phospho-binding domain. Upon phosphorylation, the probe changes conformation, which results in a change of the distance or orientation between CFP and YFP, leading to a change in FRET efficiency (Fig 1). Several probes have been published during the last decade, monitoring the activity balance of multiple kinases and phosphatases, including reporters of PKA2, PKB3, PKC4, PKD5, ERK6, JNK7, Cdk18, Aurora B9 and Plk19. Given the modular design, additional probes are likely to emerge in the near future10. 
Progression through the cell cycle is affected by stress signaling pathways 11. Notably, the cell cycle is regulated differently during unperturbed growth compared to when cells are recovering from stress12.Time-lapse imaging of cells through the cell cycle therefore requires particular caution. This becomes a problem particularly when employing ratiometric imaging, since two images with a high signal to noise ratio are required to correctly interpret the results. Ratiometric FRET imaging of cell cycle dependent changes in kinase and phosphatase activities has predominately been restricted to sub-sections of the cell cycle8,9,13,14.
Here, we discuss a method to monitor FRET-based probes using ratiometric imaging throughout the human cell cycle. The method relies on equipment that is available to many researchers in life sciences and does not require expert knowledge of microscopy or image processing.

FRET-based reporters are increasingly used to monitor kinase and phosphatase activities in live cells. Here we describe a method on how to use FRET-based reporters to assess cell cycle-dependent changes in target phosphorylation.

F&ouml;rster resonance energy transfer (FRET)-based reporters1 allow the assessment of endogenous kinase and phosphatase activities in living cells. Such ...

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 ...

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

Fluorescent cell tracking dyes, in combination with flow and image cytometry, are powerful tools with which to study the interactions and fates of different cell types in vitro and in vivo.1-5 Although there are literally thousands of publications using such dyes, some of the most commonly encountered cell tracking applications include monitoring of:
<ol>
	<li>stem and progenitor cell quiescence, proliferation and/or differentiation6-8</li>
	<li>antigen-driven membrane transfer9 and/or precursor cell proliferation3,4,10-18 and</li>
	<li>immune regulatory and effector cell function1,18-21.</li>
</ol>
Commercially available cell tracking dyes vary widely in their chemistries and fluorescence properties but the great majority fall into one of two classes based on their mechanism of cell labeling. "Membrane dyes", typified by PKH26, are highly lipophilic dyes that partition stably but non-covalently into cell membranes1,2,11. "Protein dyes", typified by CFSE, are amino-reactive dyes that form stable covalent bonds with cell proteins4,16,18. Each class has its own advantages and limitations. The key to their successful use, particularly in multicolor studies where multiple dyes are used to track different cell types, is therefore to understand the critical issues enabling optimal use of each class2-4,16,18,24.
The protocols included here highlight three common causes of poor or variable results when using cell-tracking dyes. These are:
<ol>
	<li>Failure to achieve bright, uniform, reproducible labeling. This is a necessary starting point for any cell tracking study but requires attention to different variables when using membrane dyes than when using protein dyes or equilibrium binding reagents such as antibodies.</li>
	<li>Suboptimal fluorochrome combinations and/or failure to include critical compensation controls. Tracking dye fluorescence is typically 102 - 103 times brighter than antibody fluorescence. It is therefore essential to verify that the presence of tracking dye does not compromise the ability to detect other probes being used.</li>
	<li>Failure to obtain a good fit with peak modeling software. Such software allows quantitative comparison of proliferative responses across different populations or stimuli based on precursor frequency or other metrics. Obtaining a good fit, however, requires exclusion of dead/dying cells that can distort dye dilution profiles and matching of the assumptions underlying the model with characteristics of the observed dye dilution profile.</li>
</ol>
Examples given here illustrate how these variables can affect results when using membrane and/or protein dyes to monitor cell proliferation.

Successful use of cell tracking dyes to monitor immune cell function and proliferation involves several critical steps. We describe methods for: 1) obtaining bright, uniform, reproducible label-ing with membrane dyes; 2) selecting fluorochromes and data acquisition conditions; and 3) choosing a model to quantify cell proliferation based on dye dilution.

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

Simultaneous Assessment of Kinship, Division Number, and Phenotype via Flow Cytometry for Hematopoietic Stem and Progenitor Cells

Few techniques can assess phenotype and fate for the same cell simultaneously. Most of the current protocols used to characterize phenotype, although able to generate large datasets, necessitate the destruction of the cell of interest, making it impossible to assess its functional fate. Heterogeneous biological differentiating systems like hematopoiesis are therefore difficult to describe. Building on cell division tracking dyes, we further developed a protocol to simultaneously determine kinship, division number, and differentiation status for many single hematopoietic progenitors. This protocol allows the assessment of the ex vivo differentiation potential of murine and human hematopoietic progenitors, isolated from various biological sources. Moreover, as it is based on flow cytometry and a limited number of reagents, it can quickly generate a large amount of data, at the single-cell level, in a relatively inexpensive manner. We also provide the analytical pipeline for single-cell analysis, combined with a robust statistical framework. As this protocol allows the linking of cell division and differentiation at the single-cell level, it can be used to quantitatively assess symmetric and asymmetric fate commitment, the balance between self-renewal and differentiation, and the number of divisions for a given commitment fate. Altogether, this protocol can be used in experimental designs aiming to unravel the biological differences between hematopoietic progenitors, from a single-cell perspective.

Simultaneous Assessment of Kinship, Division Number, and Phenotype via Flow Cytometry for Hematopoietic Stem and Progenitor Cells

Presented here is a flow cytometry-based technique that allows for simultaneously measuring the number of cell divisions, surface cell phenotype, and cellular kinship. Those properties can be tested statistically using a permutation-based framework.

Few techniques can assess phenotype and fate for the same cell simultaneously. Most of the current protocols used to characterize phenotype, although able ...

Immunology and Infection

Cell Cycle Analysis: Assessing CD4 and CD8 T Cell Proliferation After Stimulation Using CFSE Staining and Flow Cytometry

Source: Perchet Thibaut1,2,3, Meunier Sylvain1,2,3, Sophie Novault4, Rachel Golub1,2,3 
1 Unit for Lymphpoiesis, Department of Immunology, Pasteur Institute, Paris, France 
2 INSERM U1223, Paris, France 
3 Universit&#233; Paris Diderot, Sorbonne Paris Cit&#233;, Cellule Pasteur, Paris, France 
4 Flow Cytometry Platfrom, Cytometry and Biomarkers UtechS, Center for Translational Science, Pasteur Institute, Paris, France
The cell cycle is a universal process of life. During the cell cycle, a cell undergoes several modifications to divide into two daughter cells. This mechanism occurs throughout an organism&#39;s life in response to its needs. Cell divisions and embryonic development produce a full organism from a single-celled zygote. During adulthood, the cell cycle is central to many critical biological processes, such as tissue repairs.
Mechanisms of cell division are tightly controlled events where the cell undergoes stepwise modifications before final division. Cells that are not yet in the cycle are described as being in the Gap 0 (G0) phase. During this stage the cell is considered quiescent. When the cell starts to cycle, four distinct phases are recognized: Gap 1 (G1), Synthesis (S), Gap 2 (G2) and Mitosis (M). G1 phase is a checkpoint for resources needed by the cell for DNA synthesis. Then, S phase occurs, and DNA replication starts, followed by the G2 interphase, another checkpoint that controls all elements necessary to for the cell to divide. Finally, the cell enters mitosis and divides into two daughter cells.
Cell division is a highly informative parameter in many different biological systems. In the field of immunology, analysis of leukocyte proliferation can indicate the mechanism of the immune response. Other domains of investigation also rely on cell cycle analysis. For instance, analysis of the cell cycle during tumor development has improved our understanding of cancer.
Many fluorescent dyes are now available for tracking cell proliferation. These dyes differ in their chemical and spectral properties. Two different classes of dyes exist: protein dyes permanently combine with protein by forming a covalent bond, and membrane dyes stably intercalate within cell membranes via strong hydrophobic associations. In vitro and in vivo studies of immune cell proliferation by flow cytometry are among the most common applications of both classes of cell tracking dyes (1, 2).
CFSE (Carboxyfluorescein succinimidyl ester) is a fluorescent dye that marks dividing cells. Initially, all the cells receive the same amount of dye; dividing cells evenly split the dye they received between their two daughter cells. Consequently, the cell cycle can be followed by the progressive decrease of dye intensity in the cells. CFSE staining is followed by conventional multi-parametric flow cytometry, a high-throughput, fluorescence-based technology that allows phenotypic and functional characterization of cells based on their degree of CFSE staining (3).
In the following experiment, we assess the proliferation of CD4+ and CD8+ T cells in-vitro, following CD3 stimulation, using CFSE staining and flow cytometry.

Cell Cycle Analysis: Assessing CD4 and CD8 T Cell Proliferation

Source: Perchet Thibaut1,2,3, Meunier Sylvain1,2,3, Sophie Novault4, Rachel Golub1,2,3
1 Unit for Lymphpoiesis, Department of Immunology, Pasteur ...

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Bromodeoxyuridine Pulse Labelling Assay: A Technique to Measure Cell Proliferation

Source: Welschinger R. et al. <a href="https://www.jove.com/t/52840">Temporal Tracking of Cell Cycle Progression Using Flow Cytometry without the Need for Synchronization.</a> J. Vis. Exp. (2015)
The video describes the stepwise pulse labeling assay of mammalian cells with bromodeoxyuridine (BrdU) for measuring cell proliferation. The BrdU uptake permits the temporal tracking of cells that were in the synthesis phase at a specific point in time, without requiring the cells to be synchronized.

Source: Welschinger R. et al. Temporal Tracking of Cell Cycle Progression Using Flow Cytometry without the Need for Synchronization. J. Vis. Exp. (2015)
...

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

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