A l i f lif ti d i i i d i t li d h llAnalysis of proliferation dynamics in primary and immortalized human cellsMatthew Shallice, Jolene Bradford, Nicholas Dolman, and Chris Langsdorf, Life Technologies, Eugene, OR, USA, 97402

RESULTSFigure 1 Fluorescent protein-based cell cycle interrogation

ABSTRACTCell proliferation and the characterization of agents that affect proliferation dynamics areextremely important areas of research A variety of fluorescent tools and techniques are Figure 3 Generational analysis of proliferating primary T-lymphocytes Figure 6 Visualizing cell cycle perturbationFigure 1. Fluorescent protein-based cell cycle interrogation

Premo™ FUCCI makes use of the ubiquitin dependent degradation of two cellcycle regulators: hCdt1 and hGeminin hCdt1 participates in the attachment of

extremely important areas of research. A variety of fluorescent tools and techniques areavailable for evaluating and quantifying the fraction of eukaryotic cells in each phase of the cellcycle. Using a variety of human cell types and fluorescent reporters we characterized cellcycle profiles and their sensitivity to antimitotic agents, compounds commonly used to treat

Figure 3. Generational analysis of proliferating primary T-lymphocytes Figure 6. Visualizing cell cycle perturbationA. Control 10uM Cytochalasin D

A. B.A.B.

cycle regulators: hCdt1 and hGeminin. hCdt1 participates in the attachment ofhelicases to the DNA origin of replication during G1 phase. hGeminin negativelyregulates cdt1; this feedback loop helps to guide the appropriate replication ofDNA during the cell cycle. Figure 1A. provides an illustration of the biphasiccycling of the geminin and Cdt1 fusions through the cell cycle.

cancer. The fraction of cells in each stage of the cell cycle was measured using a DNA bindingdye in conjunction with a fluorescent protein ubiquitination cell cycle indicator expressed bytransfecting cells. Synthesis of DNA during S-phase was detected using a click1,2 reaction tofluorescently label 5-ethynyl-2’-deoxyuridine (EdU) a thymidine analog incorporated into ou

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Human Cervical Carcinoma (HeLa)

cycling of the geminin and Cdt1 fusions through the cell cycle.fluorescently label 5 ethynyl 2 deoxyuridine (EdU), a thymidine analog incorporated intoreplicating DNA. Generational analysis was performed using a novel violet-excitable cellproliferation tracking dye4,5,6. A comparison of primary human cells and immortalized humancells derived from tumors (cervical carcinoma, alveolar epithelium, and osteosarcoma)d t t d th t i t li d ll h i d iti it t i t b l t b ti

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A549 cells Untreated HeLa cells HeLa cells + 200 nM Taxoldemonstrated that immortalized cells have an increased sensitivity to microtubule perturbation.Moreover non-immortalized cells show a significantly enriched G0 population compared toimmortalized cells, reflecting a decreased proliferative index.

INTRODUCTION

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INTRODUCTIONHere we present an overview of several techniques used to characterize the growth of primaryand immortalized human cells. One technique uses the Fluorescence Ubiquitination Cell Cycle

CellTrace™ Violet FluorescenceHuman PBMCs, isolated from whole blood using a Ficoll-Paque™ Plus density gradient, were stained with 10 µMCellTrace™ Violet, stimulated with mouse anti-human CD3 and IL-2, and grown in culture for seven days. SYTOX®

AADvanced™ Dead Cell Stain and mouse anti-human CD4-Alexa Fluor® 488 were used to identify live CD4+ cells. A

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Indicator, Premo™ FUCCI3. This is a fluorescent protein-based sensor that employs a RedFluorescent Protein (RFP) and a Green Fluorescent Protein (GFP); each of which is fused toone of two different regulators of the cell cycle: Cdt1 and geminin. Ubiquitin E3 ligases addubiquitin to the Cdt1–RFP and geminin–GFP fusions, thereby targeting these proteins to the

gating strategy was employed to eliminate debris and doublets from analysis. One million total cells were collected andanalyzed on the Attune® Acoustic Flow Cytometer. Figure A is a fluorescence histogram of CellTrace™ Violetfluorescence, with each peak corresponding to a discrete generation of proliferating cells (Purple overlay representsunstimulated PBMCs) Figure B displays the same data analyzed with the algorithms in ModFit LT™ software to identifyindividual generations.

Human cervical carcinoma and human corneal epithelial cells were treated with 10 µM Cytochalasin D for 16 hours or leftuntreated. Microtubules were visualized with anti-α tubulin goat anti-mouse Alexa Fluor® 555. Actin was labeled with AlexaFxCycle™ Violet FluorescenceFxCycle™ Violet Fluorescence FxCycle™ Violet Fluorescence

Figure 4. Multiparameter proliferation analysis of stimulated primary T-lymphocytesFigure 1. Cell cycle phase characterization using flow cytometry with Premo™ FUCCI . Alveolar epithelial (A549, Figure1B) and cervival carcinoma (HeLa, Figure 1C) cells were transduced with Premo™ FUCCI constructs at a multiplicity ofinfection (MOI) of 20. Cells were stained with the DNA content dye FxCycle™ Violet and the viability dye LIVE/DEAD®

Fixable Far Red Dead Cell Stain. HeLa cells were treated with 200 nM Taxol for 24 hours (Figure 1D). Cells were

ubiquitin to the Cdt1 RFP and geminin GFP fusions, thereby targeting these proteins to theproteasome for degradation. Temporal regulation of E3 ligase activity results in the biphasiccycling of the geminin and Cdt1 fusions through the cell cycle3. Another technique uses flowcytometry to measure DNA content using fluorescent DNA-selective stains that exhibit

i i i l ti l t DNA Fl t t i l i f th t i d

individual generations. u ea ed c o ubu es e e sua ed a α ubu goa a ouse e a uo 555 c as abe ed e aFluor® 488 Phalloidin. Nuclei were counterstained with Hoescht 33342. Untreated control cells (A and C) appearunaffected as evidenced by the intact actin filaments (green) and microtubules (red). Cells treated with Cytochalasin D (Band D) have intact microtubules (red), but have disrupted actin filaments and greatly diminished actin polymerization(green) resulting in cell cycle arrest at the transition from G1 to S phase. Images created with an Applied PrecisionDeltaVision® Core microscope with standard DAPI FITC and TRITC filters

FxCycle Violet FluorescenceFxCycle Violet Fluorescence FxCycle Violet Fluorescence

B.A.( g )analyzed on a BD™ LSRII flow cytometer. Overlays of FxCycle™ Violet stain with GFP and RFP expression demonstratethe correlation of DNA content with hGeminin-emGFP & hCdt1-tagRFP expression. The G1 (2N) population expresseshCdt1-tagRFP whereas G2/M (4N) population expresses hGeminin-emGFP. The S-phase population of cells expresses anincreasing proportion of hGeminin-emGFP expressing cells and a decreasing proportion of hCdt1-tagRFP positive cells.The Taxol treated HeLa cells show a significant increase in the G2/M phase (62%) compared to the untreated HeLa cells

emission signals proportional to DNA mass. Flow cytometric analysis of these stainedpopulations is then used to produce a frequency histogram that reveals the various phases ofthe cell cycle. Cells in S-phase of the cell cycle can be more accurately identified by detectingthe incorporation of the thymidine analog EdU into newly synthesized DNA using a Click-iT®

CONCLUSIONS

DeltaVision Core microscope with standard DAPI, FITC, and TRITC filters.

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The Taxol treated HeLa cells show a significant increase in the G2/M phase (62%) compared to the untreated HeLa cells(11%) as determined using ModFit LT™ modeling software (inset plots in figures 1C and 1D illustrate the modeled resultsshowing the three overlapping phase distributions).

Figure 2 Multiparameter Analysis of Cell Cycle Perturbation

p y g y y glabeling reaction1,2. Multigenerational proliferation can be analyzed using the cell proliferationdye CellTrace™ Violet. When cells such as primary T-lymphocytes are stained withCellTrace™ Violet, grown in culture, and evaluated on a flow cytometer, the resultingfluorescence histogram contains several peaks each of which corresponds to a discrete

Cell-cycle information provided by the fluorescent protein-based Premo™ FUCCI Cell CycleSensor correlates well with flow cytometry data provided by the nucleic acid binding dyeFxCycle™ Violet.r®

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Figure 2. Multiparameter Analysis of Cell Cycle Perturbationfluorescence histogram contains several peaks, each of which corresponds to a discretegeneration of proliferating cells4,5,6.

MATERIALS AND METHODS

FxCycle Violet.

Cell cycle disruption due to microtubule perturbation can be quantified using a dual-parameterplot to combine the DNA content information from FxCycle™ Violet with the S-phase analysisprovided by Click-iT® EdU.

Human Alveolar Epithelial (A549) Human Cervical Carcinoma (HeLa) Human Primary Dermal Fibroblast (hDFa)

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G0/G1/G2/M phases

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Human alveolar epithelial cells (A549) and cervical carcinoma cells (HeLa) were transducedwith Premo™ FUCCI constructs at a multiplicity of infection (MOI) of 20. Cells were stainedwith the DNA content dye FxCycle™ Violet and the viability dye LIVE/DEAD® Fixable Far RedDead Cell Stain HeLa cells were also treated with 200 nM Taxol for 24 hours (Figure 1)

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Individual generations of proliferating lymphocytes can be identified using a histogram ofCellTrace™ Violet fluorescence intensity. Further information of proliferation dynamics can beobtained by combining CellTrace™ Violet with Click-iT® EdU to identify the proportion of cells

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Proliferating human lymphocytes were stained with 10 µM CellTrace™ Violet and grown in culture for seven days. Cellswere stained with FxCycle™ Far Red to evaluate DNA content. Click-iT® EdU Alexa Fluor® 488 labeling was used toidentify cells in S phase of the cell cycle Figure A displays the DNA content on the X axis and the progression of

Dead Cell Stain. HeLa cells were also treated with 200 nM Taxol for 24 hours . (Figure 1)Primary Human Dermal Fibroblasts, cervical carcinoma cells (HeLa), and human alveolarepithelial cells (A549) were treated with 500 nM Taxol for 24 hours followed by a 10 µM pulseof EdU 2 hours prior to analysis Cells were fixed permeabilized and stained with the DNA

in each generation that have recently synthesized DNA. CellTrace™ Violet can also becombined with a DNA content dye such as FxCycle™ Far Red to distinguish quiescent cellsin each generation from those that are actively proceeding through cell division.

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identify cells in S phase of the cell cycle. Figure A displays the DNA content on the X-axis and the progression ofgenerations on the Y-axis. The parent generation of cells is at the top of the plot (pink). Figure B is a dual-parametercontour plot showing s-phase analysis as indicated by Click-iT® EdU Alexa Fluor® 488 fluorescence on the y-axis. Theparent generation of cells can be seen on the far right of the plot, with successive generations represented by clusters ofdots to the left of the initial generation. Cells from each generation which are actively synthesizing DNA are seen at the topf th fi

of EdU 2 hours prior to analysis. Cells were fixed, permeabilized, and stained with the DNAcontent dye FxCycle™ Violet. (Figure 2)Human peripheral blood mononuclear cells (PBMCs) were isolated from whole blood, stainedwith 10 µM CellTrace™ Violet, and resuspended in OpTmizer™ T-Cell Expansion Buffer

Drug-induced cell cycle arrest can be visualized in primary and immortalized cells byfluorescently labeling microtubules and actin filaments.

FxCycle™ Violet Fluorescence FxCycle™ Violet FluorescenceFxCycle™ Violet Fluorescence

ED F of the figure.with 10 µM CellTrace Violet, and resuspended in OpTmizer T Cell Expansion Buffercontaining L-glutamine, penicillin and streptomycin. Cell proliferation was stimulated with 200ng mouse anti-human CD3 (clone S4.1) and 100 ng Interleukin-2 (IL-2) per milliliter cells . After7 days in culture, an aliquot of cells was fed a 2 hour pulse of EdU, and then labeled usingCli k iT® EdU Al Fl ® 488 d 1 2 M ti h CD4 R h th i d

REFERENCES

1 Ch bi h 4 1147 (2003)500 nM Taxol Fluo

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Figure 5. Evaluation of sensitivity to microtubule perturbationClick-iT® EdU Alexa Fluor® 488 dye1,2. Mouse anti-human CD4 R-phycoerythrin andLIVE/DEAD® Fixable Yellow Dead Cell Stain were used to gate on live CD4+ lymphocytes.The DNA content dye FxCycle™ Far Red was applied 30 minutes prior to analysis. Cells wereanalyzed on a Becton Dickinson (BD™) LSRII flow cytometer and proliferation analysis was

1. Chembiochem 4, 1147 (2003)

2. J Am Chem Soc 125, 3192 (2003)

3. Sakaue-Sawano et al. (2008) Cell 132:487–498.

500 nM Taxol

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performed with ModFit LT™ Software (Verity Software House). Cells were also analyzed onthe Attune® Acoustic Cytometer without EdU incorporation and using mouse anti-human CD4-Alexa Fluor® 488 with SYTOX® AADvanced™ dead cell stain to gate on live CD4+lymphocytes (Figures 3&4)

4. Wallace, et al. Cytometry Part A 73A: 1019-1034, 2008.

5. Lyons AB, Parish CR. Journal of Immunological Methods 1994; 171:131-37.

6. Givan AL, et al. Journal of Immunological Methods 1999; 230:99-112.FxCycle™ Violet Fluorescence FxCycle™ Violet FluorescenceFxCycle™ Violet Fluorescence

152025

EdU positivelymphocytes. (Figures 3&4)

A549 cells were either untreated or treated with 500 nM Nocodazole for 16 hours, followed bya 30 minute pulse of 10 µM EdU. Cells were fixed with 4% paraformaldehyde andpermeabilized with 0 1% Triton® X-100 in PBS Incorporated EdU was detected with Click-iT®

TRADEMARKS/LICENSINGPrimary Human Dermal Fibroblasts, cervical carcinoma cells (HeLa), and human alveolar epithelial cells (A549) weregrown in culture and given a 500 nM pulse of the microtubule-stabilizing drug Taxol. A 10 µM pulse of EdU was fed twohours prior to analysis. Incorporated EdU was detected using a reaction with Click-iT® EdU Alexa Fluor® 488 dye azide. 0

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permeabilized with 0.1% Triton X 100 in PBS. Incorporated EdU was detected with Click iTEdU Alexa Fluor® 488 azide, α tubulin was detected with anti α tubulin-Goat anti-Mouse AlexaFluor® 555, and nuclei were counterstained with Hoechst 33342. (Figure 5)

Human cervical carcinoma and human corneal epithelial cells were treated with 10 µM

For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.© 2010 Life Technologies Corporation. All rights reserved. The trademarks mentioned hereinare the property of Life Technologies Corporation or their respective owners ModFit LT™ is

Cells were analyzed using an Attune® Acoustic Focusing Cytometer with 405 nm and 488 nm lasers using standard VL1(450/40) and BL1 (530/30) emission filters. Dual parameter plots of FxCycle™ Violet vs. Alexa Fluor® 488 Click-iT® EdUfluorescence can be used to clearly identify cells in different phases of the cell cycle. Untreated A549 cells have asignificantly higher percentage of cells in S-phase and lower percentage in G0/G1 than other cells types, reflecting a higherproliferative index. Upon treatment with Taxol, more than 90% of A549 and HeLa cells were arrested in G2 phase as their

Human alveolar epithelial cells (A549) were treated with 50 nM Nocodazole for 16 hours, followed by a 30 minute pulse of10 µM EdU. A. Cells were labeled with Click-iT® EdU Alexa Fluor® 488 azide (green). α tubulin was detected with anti α-tubulin goat anti-mouse Alexa Fluor® 555 (red). Nuclei were counterstained with Hoechst 33342 (blue). B. Approximately33% of untreated cells were in S-phase, compared to only 12% of Nocodazole treated cells. The decrease results from

Control 500nM Nocodazole

p µCytochalasin D for 16 hours or left untreated. Microtubules were visualized with anti-α tubulingoat anti-mouse Alexa Fluor® 555. Actin was labeled with Alexa Fluor® 488 Phalloidin. Nucleiwere counterstained with Hoescht 33342. (Figure 6)

are the property of Life Technologies Corporation or their respective owners. ModFit LT™ isa trademark of Verity Software House. Triton® is a trademark of Union Carbide Corporation.

p p , 2 pmicrotubules were prevented from depolymerizing prior to cell division. A similar pattern of arrest was seen with primaryhuman dermal fibroblasts, with the exception of approximately 29% of cells which remain in the quiescent state of G0.

33% of untreated cells were in S phase, compared to only 12% of Nocodazole treated cells. The decrease results frommicrotubule depolymerization and mitotic arrest, preventing entry into S-phase. Images created with an Applied PrecisionDeltaVision ® microscope with standard DAPI, FITC, and TRITC filters.

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