Dynamic Proliferation Assessment in Flow Cytometry

Simone Diermeier‐Daucher1, Gero Brockhoff1

1 Department of Gynaecology and Obstetrics, University of Regensburg, Regensburg, Germany
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 8.6
DOI:  10.1002/0471143030.cb0806s48
Online Posting Date:  September, 2010
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Abstract

Dynamic proliferation assessment via flow cytometry is legitimately supposed to be the most powerful tool for recording cell cycle kinetics in‐vitro. The preeminent feature is a single cell–based multi‐informative analysis by temporal high‐resolution. Flow cytometric approaches are based on labeling of proliferating cells via thymidine substitution by a base analog (e.g., 5‐bromo‐2′‐deoxyuridine, BrdU) that is added to cell cultures either for a short period of time (pulse labeling) or continuously until cell harvesting. This unit describes the alternative use of the thymidine analog 5‐ethynyl‐2′‐deoxyuridine (EdU) in place of BrdU for three different applications: (1) dynamic proliferation assessment by EdU pulse cell labeling; (2) the same approach as (1) but in combination with live/dead cell discrimination; and (3) dynamic cell cycle analysis based on continuous cell labeling with EdU and Hoechst fluorochrome quenching. In contrast to the detection of BrdU incorporation, EdU‐positive cells can be identified by taking advantage of click chemistry, which facilitates a simplified and fast cell preparation. Further analysis options but also limitations of the utilization of EdU are discussed. Curr. Protoc. Cell Biol. 48:8.6.1‐8.6.23. © 2010 by John Wiley & Sons, Inc.

Keywords: cell proliferation; EdU; flow cytometry; cell cycle kinetics

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Dynamic Cell Proliferation Assessment with EdU Cell Labeling and Detection by Click Chemistry
  • Alternate Protocol 1: Dynamic Cell Proliferation Assessment with EdU Cell Labeling and Detection by Click Chemistry in Combination with Live/Dead Discrimination
  • Support Protocol 1: Evaluation of Cell Death by Determination of the Sub‐G1 Fraction
  • Basic Protocol 2: Dynamic Cell Proliferation Assessment by Edu/Hoechst Quenching
  • Support Protocol 2: Quantification of the G0 Cell Fraction with the Edu/Hoechst Quenching Technique
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Dynamic Cell Proliferation Assessment with EdU Cell Labeling and Detection by Click Chemistry

  Materials
  • Click‐iT EdU Alexa Fluor 488 Cell Proliferation Assay Kit (store kit at 2°–6°C, do not freeze):
    • 5‐Ethynyl‐2′‐deoxyuridine (EdU)
    • DMSO
    • Click‐iT fixative
    • 10× saponin‐based permeabilization and wash reagent
    • Alexa Fluor 488 azide
    • 10× Click‐iT EdU reaction buffer
    • 100 mM CuSO 4 stock solution
    • Click‐iT EdU buffer additive
    • Click‐iT EdU cell cycle dye 633
    • 20 mg/ml RNase A
  • PBS/1% BSA: phosphate‐buffered saline (PBS; appendix 2A) containing 1% bovine serum albumin (BSA); store up to 2 weeks at 4°C
  • BT474 breast cancer cells (ATCC #HTB‐20; see unit 1.1 for culture of mammalian cells)
  • Complete Dulbecco's modified Eagle medium (DMEM; unit 1.1)
  • Complete DMEM containing 5% fetal bovine serum (FBS; DMEM‐5; unit 1.1)
  • Complete DMEM containing 2% FBS (DMEM‐2)
  • Drug to be tested for its effect on cell proliferation (e.g., trastuzumab, also known as rhuMAb, 4D5, and herceptin, humanized monoclonal antibody acting on the ErbB2 (HER2/neu) receptor)
  • Trypsin/EDTA solution ( appendix 2A)
  • PBS/0.01% NaN 3/0.2% BSA: phosphate‐buffered saline (PBS; appendix 2A) containing 0.01% NaN 3 and 0.2% BSA; store up to 2 weeks at 4°C
  • 75‐cm2 cell culture flasks
  • 15‐ and 50‐ml centrifuge tubes (e.g., polypropylene tubes, Greiner Bio‐one)
  • Refrigerated centrifuge, 4°C
  • Water jet vacuum pump (e.g., BRAND), optional
  • Hemacytometer ( appendix 3B)
  • 5‐ml sample tubes (e.g., 12 × 75–mm polystyrene round‐bottomed tubes, BD)
  • Flow cytometer with 488‐nm and 633‐nm excitation lasers (see Table 8.6.3 for required/appropriate filters)
  • Acquisition software that stores data in list‐mode flow cytometry standard (FCS) files (e.g., BD FACS Diva software v6.1.1, BD Biosciences)
  • Evaluation software (e.g., FlowJo 7.6.1, Tree star)
NOTE: Protect labeling solutions and labeled samples from light.

Alternate Protocol 1: Dynamic Cell Proliferation Assessment with EdU Cell Labeling and Detection by Click Chemistry in Combination with Live/Dead Discrimination

  • LIVE/DEAD Fixable Violet Dead Cell Stain Kit for 405‐nm excitation (Molecular Probes/Invitrogen)
  • Drug for induction of cell death in the cell line of interest, e.g., sodium azide (NaN 3; Sigma) for BT474 (Diermeier‐Daucher et al., )
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • 4% (w/v) paraformaldehyde (PFA; see recipe)
NOTE: Protect labeling solutions and labeled samples from light.

Support Protocol 1: Evaluation of Cell Death by Determination of the Sub‐G1 Fraction

  Materials
  • BT474 breast cancer cells (ATCC #HTB‐20; see unit 1.1 for culture of mammalian cells)
  • Complete DMEM containing 5% (v/v) fetal bovine serum (FBS; DMEM‐5; unit 1.1)
  • Drug to be tested for its effect on cell proliferation (e.g., trastuzumab, also known as rhuMAb, 4D5, and herceptin, humanized monoclonal antibody acting on the ErbB2 (HER2/neu) receptor)
  • Complete DMEM containing 2% (v/v) FBS (DMEM‐2), optional
  • 10 mM 5‐ethynyl‐2′‐deoxyuridine (EdU; e.g., Molecular Probes/Invitrogen) in DMSO
  • Trypsin/EDTA solution ( appendix 2A)
  • PBS/0.01% NaN 3/0.2% BSA: phosphate‐buffered saline (PBS; appendix 2A) containing 0.01% (w/v) NaN 3 and 0.2% (w/v) BSA; store up to 2 weeks at 4°C
  • Freezing medium without phenol red (e.g., RPMI, 10% FBS, 10% DMSO; Brockhoff, ; see unit 1.1), optional
  • DNA washing buffer (see recipe)
  • DNA staining buffer (see recipe)
  • 250 µg/ml propidium iodide (PI; e.g., Sigma) solution in PBS
  • 75‐cm2 culture flasks
  • 50‐ml centrifuge tubes (e.g., Polypropylene tubes, Greiner Bio‐one)
  • Refrigerated centrifuge, 4°C
  • Hemacytometer (unit 1.1)
  • Water jet vacuum pump (e.g., BRAND), optional
  • 37°C water bath (optional)
  • 5‐ml sample tubes (e.g., 12 × 75–mm polystyrene round‐bottomed tubes, BD)
  • 70‐µm nylon mesh, optional
  • Flow cytometer with a 488‐nm and UV laser excitation (see Table 8.6.3 for required filters)
  • Acquisition software that stores data in list‐mode flow cytometry standard (FCS) files (e.g., BD FACS Diva software V6.1.1, BD Biosciences)
  • Evaluation software (e.g., FlowJo 7.6.1, Tree star)
NOTE: Protect labeling solutions and labeled samples from light.
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Figures

Videos

Literature Cited

   Bradford, J.A. and Buller, G.M. 2008. Dead cell stains in flow cytometry: A comprehensive analysis. XXIV International Congress, Program and Abstracts, 417/P315:247.
   Bradford, J.A., Clarke, S.T., Buck, S.B., Hill, D., Chen, Y.‐T., Gee, K., Agnew, B., and Salic, A. 2008a. Detection of S‐phase cell cycle progression using 5‐ethynyl‐2′‐deoxyuridine incorporation with click chemistry. XXIV International Congress, Program and Abstracts, 279/P177:197‐198.
   Bradford, J.A., Clarke, S.T., Hill, D., and Chen, Y‐T. 2008b. Characterization of DNA content, cyclin B1 and phosphorylated histone H3 with direct S‐phase using EdU incorporation in multiparameter testing of cell lines with cell cycle blocking agents. XXIV International Congress, Program and Abstracts, 280/P178:198.
   Bradford, J.A., Hill, D.M., Clarke, S.T., and Chen, Y.‐T. 2008c. Detection of DNA synthesis by automated microscopy and image analysis: Comparison of BrdU method and a new click chemistry based EdU method. XXIV International Congress, Program and Abstracts, 283/P181:199.
   Brockhoff, G. 2009. Flow cytometric DNA und proliferation analysis. In Cellular Diagnostics—Basic Principles, Methods and Clinical Applications in Flow Cytometry. (U. Sack and G. Rothe, eds.) pp. 390‐425. Karger Publishing, Unionville, CT.
   Brockhoff, G., Heiss, P., Schlegel, J., Hofstaedter, F., and Knuechel, R. 2001. Epidermal growth factor receptor, c‐erbB2 and c‐erbB3 receptor interaction, and related cell cycle kinetics of SK‐BR‐3 and BT474 breast carcinoma cells. Cytometry 44:338‐348.
   Brockhoff, G., Heckel, B., Schmidt‐Bruecken, E., Plander, M., Hofstaedter, F., Vollmann, A., and Diermeier, S. 2007. Differential impact of cetuximab, pertuzumab and trastuzumab on BT474 and SK‐BR‐3 breast cancer cell proliferation. Cell Prolif. 40:488‐507.
   Buck, S.B., Bradford, J., Gee, K.R., Agnew, B.J., Clarke, S.T., and Salic, A. 2008. Detection of S‐phase cell cycle progression using 5‐ethynyl‐2′‐deoxyuridine incorporation with click chemistry, an alternative to using 5‐bromo‐2′‐deoxyuridine antibodies. Biotechniques. 44:927‐929.
   Cappella, P., Gasparri, F., Pulici, M., and Moll, J. 2008a. A novel method based on click chemistry, which overcomes limitations of cell cycle analysis by classical determination of BrdU incorporation, allowing multiplex antibody staining. Cytometry A 73:626‐636.
   Cappella, P., Gasparri, F., Pulici, M., and Moll, J. 2008b. Cell proliferation method: Click chemistry based on BrdU coupling for multiplex antibody staining. Curr. Protoc. Cytom. 45:7.34.1‐7.34.13.
   Clarke, S.T., Bradford, J.A., and Godfrey, B. 2008. Dual pulse labeling of S‐phase populations using click chemistry. DGfZ‐ESCCA joint abstracts from the 18th annual meeting of the German Society of Cytometry (DGfZ) and the 8th Euroconference of Clinical Cell Analysis by the European Society for Clinical Cell Analysis (ESCCA). Cytometry B Clin. Cytom. 74B:378‐415.
   Darzynkiewicz, Z. and Juan, G. 1997. Analysis of DNA content and BrdU incorporation. Curr. Protoc. Cytom. 2:7.7.1‐7.7.9.
   Diermeier‐Daucher, S., Schmidt‐Bruecken, E., Kubbies, M., Kunz‐Schughart, L.A., and Brockhoff, G. 2004. Continuous bromodeoxyuridine (BrdU) exposition differentially affects cell cycle progression of human breast and bladder cancer cell lines. Cell Prolif. 37:195‐206.
   Diermeier‐Daucher, S., Horvàth, G., Knuechel‐Clarke, R., Hofstaedter, F., Szöllõsi, J., and Brockhoff, G. 2005. Epidermal growth factor receptor coexpression modulates susceptibility to herceptin in HER2/neu overexpressing breast cancer cells via specific erbB‐receptor interaction and activation. Exp. Cell Res. 304:604‐619.
   Diermeier‐Daucher, S., Clarke, S.T., Hill, D., Vollmann‐Zwerenz, A., Bradford, J.A., and Brockhoff, G. 2009. Cell type specific applicability of 5‐ethynyl‐2′‐deoxyuridine (EdU) for dynamic proliferation assessment in flow cytometry. Cytometry A 75:535‐546.
   Endl, E., Steinbach, P., Knüchel, R., and Hofstädter, F. 1997. Analysis of cell cycle‐related Ki‐67 and p120 expression by flow cytometric BrdUrd‐Hoechst/7AAD and immunolabeling technique. Cytometry 29:233‐241.
   George, T.C., Bradford, J.A., Clarke, S.T., Godfrey, W.L., Kong, R., Hall, B.E., and Morrissey, P.M. 2008. Effect of p27KIP1 nuclear localization on cell cycle progression and apoptotic changes as measured using ImageStream imaging cytometry of cells labeled with Click‐iT EdU and Vybrant DyeCycle stains. XXIV International Congress, Program and Abstracts. 20:106.
   Kubbies, M., Schindler, D., Hoehn, J., and Rabinovitch, P.S. 1985. BrdU‐Hoechst flow cytometry reveals regulation of human lymphocyte growth by donor age‐related growth fraction and transition rate. J. Cell. Physiol. 125:229‐234.
   Latt, S.A., George, Y.S., and Gray, J.W. 1977. Flow cytometric analysis of bromodeoxyuridine‐substituted cells stained with 33258 Hoechst. J. Histochem. Cytochem. 25:927‐934.
   Poot, M., Schindler, D., Kubbies, M., Hoehn, H., and Rabinovitch, P.S. 1988. Bromodeoxyuridine amplifies the inhibitory effect of oxygen on cell proliferation. Cytometry. 9:332‐338.
   Poot, M., Schuster, A., and Hoehn, H. 1991. Cytostatic synergism between bromodeoxyuridine, bleomycin, cisplatin and chlorambucil demonstrated by a sensitive cell kinetic assay. Biochem. Pharmacol. 41:1903‐1909.
   Poot, M., Hoehn, H., Kubbies, M., Grossmann, A., Chen, Y., and Rabinovitch, P.S. 1994. Cell‐cycle analysis using continuous bromodeoxyuridine labeling and Hoechst 33358‐ethidium bromide bivariate flow cytometry. Methods Cell. Biol. 41:327‐340.
   Poot, M., Rosato, M., and Rabinovitch, P.S. 2001. Analysis of cell proliferation and cell survival by continuous BrdU labeling and multivariate flow cytometry. Curr. Protoc. Cytom. 15:7.14.1‐7.14.9.
   Rabinovitch, P.S., Kubbies, M., Chen, Y.C., Schindler, D., and Hoehn, H. 1988. BrdU‐Hoechst flow cytometry: A unique tool for quantitative cell cycle analysis. Exp. Cell Res. 174:309‐318.
   Roederer, M. 2002. Compensation in flow cytometry. Curr. Protoc. Cytom. 22:1.14.1‐1.14.20.
   Rostovtsev, V.V., Green, L.G., Fokin, V.V., and Sharpless, K.B. 2002. A stepwise huisgen cycloaddition process: Copper(I)‐catalyzed regioselective “ligation” of azides and terminal alkynes. Angew. Chem. Int. Ed. Engl. 41:2596‐2599.
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Internet Resources
   http://probes.invitrogen.com/media/pis/mp34955.pdf
  Available dyes for live/dead discrimination with the LIVE/DEAD dead cell stain kit.
   http://www.invitrogen.com/site/us/en/home/brands/Molecular‐Probes/Key‐Molecular‐Probes‐Products/Click‐iT‐Detection‐Assays/click‐iT‐Edu.html#flow
  Available dyes for EdU labeling with Click‐iT EdU Cell Proliferation Assays.
   http://www.isac‐net.org/congress2008/documents/colorbars_isac_program.pdf
  XXIV International Congress 2008, program and abstracts.
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