Flow Cytometry‐Based Quantification of Cell Proliferation in the Mixed Cell Co‐Culture

Bogdan I. Gerashchenko1, Roger W. Howell2

1 Department of Radiobiology and Ecology, R.E. Kavetsky Institute of Experimental Pathology, Oncology and Radiobiology, Kyiv, Ukraine, 2 Department of Radiology, UMDNJ−New Jersey Medical School Cancer Center, Newark, New Jersey
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 9.40
DOI:  10.1002/0471142956.cy0940s63
Online Posting Date:  January, 2013
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Abstract

In cell communities, among the crucial signals that govern cell function are those generated locally by surrounding cells. A co‐culture of mixed homotypic or heterotypic cells, which is often used in various fields of experimental biology and medicine, can be applied for elucidation of the role of cell proximity in modulating proliferative responses. Quick and reliable quantification of the changes in proliferation of each of the mixed cell populations as a result of their co‐culture is of importance. For this purpose, flow cytometry together with fluorescent tracers that do not affect cell proliferation can be used. Curr. Protoc. Cytom. 63:9.40.1‐9.40.10. © 2013 by John Wiley & Sons, Inc.

Keywords: cell proliferation; cell proximity; mixed co‐culture; fluorescent cell tracers; flow cytometry; proliferation ratio

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Flow Cytometry‐Based Quantification of Cell Proliferation in the Mixed Cell Co‐Culture
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Flow Cytometry‐Based Quantification of Cell Proliferation in the Mixed Cell Co‐Culture

  Materials
  • 1 mM 1,1′‐dioctadecyl‐3,3,3′3′‐tetramethylindocarbocyanine perchlorate (DiI; see recipe)
  • 10 mM 5‐(and ‐6)‐carboxyfluorescein diacetate succinimidyl ester (CFDA SE; see recipe)
  • Culture medium, 37°C
  • Monolayer of cells for homotypic or heterotypic cell co‐culture model
  • Phosphate‐buffered saline (PBS; appendix 2A), 37°C
  • 0.05% Trypsin/0.53 mM EDTA
  • 37°C, CO 2 humidified incubator
  • 12 × 75–mm polystyrene tubes
  • Microscope (phase contrast)
  • Hemacytometer or automatic cell counter
  • 60 × 15–mm Petri dishes
  • Refrigerated centrifuge with swinging buckets and 12 × 75–mm tube carriers
  • Flow cytometer equipped with an argon‐ion laser (488‐nm)
  • Software for analyzing data obtained by flow cytometry (e.g., CELLQuest software from Becton‐Dickinson Immunocytometry Systems)
  • Additional reagents and equipment for cell culture ( appendix 3B)
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Figures

Videos

Literature Cited

Literature Cited
   Burke, J.M. 1983. Cell‐cell contact promotes DNA synthesis in retinal glia but not in fibroblasts. Exp. Cell Res. 146:204‐206.
   Burke, J.M. and Foster, S.J. 1985. Induction of DNA synthesis by co‐culture of retinal glia and pigment epithelium. Invest. Ophthalmol. Vis. Sci. 26:636‐642.
   Gerashchenko, B.I. and Howell, R.W. 2003a. Flow cytometry as a strategy to study radiation‐induced bystander effects in co‐culture systems. Cytometry A 54:1‐7.
   Gerashchenko, B.I. and Howell, R.W. 2003b. Cell proximity is a prerequisite for the proliferative response of bystander cells co‐cultured with cells irradiated with γ‐rays. Cytometry A 56:71‐80.
   Gerashchenko, B.I. 2008. Quantitative assessment of cell proliferation in the co‐culture of mixed cell populations by flow cytometry. Cytometry A 73:492‐493.
   Krtolica, A., Parrinello, S., Lockett, S., Desprez, P.‐Y., and Campisi, J. 2001. Senescent fibroblasts promote epithelial cell growth and tumorigenesis: A link between cancer and aging. Proc. Natl. Acad. Sci. U.S.A. 98:12072‐12077.
   Krtolica, A., de Solorzano, C.O., Lockett, S., and Campisi, J. 2002. Quantification of epithelial cells in coculture with fibroblasts by fluorescence image analysis. Cytometry 49:73‐82.
   Saeki, T., Morioka, T., Arakawa, M., Shimizu, F., and Oite, T. 1991. Modulation of mesangial cell proliferation by endothelial cells in co‐culture. Am. J. Pathol. 139:949‐957.
   Spink, B.C., Cole, R.W., Katz, B.H., Gierthy, J.F., Bradley, L.M., and Spink, D.C. 2006. Inhibition of MCF‐7 breast cancer cell proliferation by MCF‐10A breast epithelial cells in co‐culture. Cell Biol. Int. 30:227‐238.
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