Flow Cytometric Analysis of Cell Division by Dilution of CFSE and Related Dyes

A. Bruce Lyons1, Stephen J. Blake2, Kathleen V. Doherty3

1 School of Medicine, The University of Tasmania, Hobart, Tasmania, Australia, 2 Diamantina Institute, The University of Queensland, Brisbane, Queensland, Australia, 3 Faculty of Health Science, The University of Tasmania, Hobart, Tasmania, Australia
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 9.11
DOI:  10.1002/0471142956.cy0911s64
Online Posting Date:  April, 2013
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The technique described in this unit uses the intracellular fluorescent label carboxyfluorescein diacetate succinimidyl ester (CFSE) to track proliferating cells. Covalently bound CFSE is divided equally between daughter cells, allowing discrimination of successive rounds of cell division. The technique is applicable to in vitro cell division, as well as to in vivo division of adoptively transferred cells and can resolve eight or more successive generations. CFSE is long lived, permitting analysis for several months after cell transfer, and has the same spectral characteristics as fluorescein, so monoclonal antibodies conjugated to phycoerythrin or other compatible fluorochromes may be used to immunophenotype the dividing cells. In addition, information is given on a second‐generation dye, Cell Trace Violet (CTV), excited by 405‐nm blue laser light. CTV is chemically related to CFSE, but allows the 488‐nm line of the Argon laser to be used for other probes. Curr. Protoc. Cytom. 64:9.11.1‐9.11.12. © 2013 by John Wiley & Sons, Inc.

Keywords: flow cytometry; cell division; CFSE; CTV; proliferation; adoptive transfer

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Table of Contents

  • Introduction
  • Basic Protocol 1: Determination of Cell Division Using Carboxyfluorescein Diacetate Succinimidyl Ester (CFDA‐SE or CFSE)
  • Alternate Protocol 1: Determination of Cell Division Using Cell Trace Violet (CTV)
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Determination of Cell Division Using Carboxyfluorescein Diacetate Succinimidyl Ester (CFDA‐SE or CFSE)

  • Single‐cell suspension of cells of interest (e.g., lymphoid cells, cultured cell line)
  • Phosphate‐buffered saline (PBS; appendix 2A)/0.1% (w/v) BSA
  • 5 mM 5‐(and ‐6)‐carboxyfluorescein diacetate succinimidyl ester (CFDA‐SE or CFSE; see recipe)
  • RPMI 1640/10% (v/v) FBS
  • Culture medium or injection medium appropriate for the experiment
  • Antibodies for immunophenotyping (optional)
  • 37°C incubator
  • Flow cytometer with 488‐nm argon laser, or multi‐laser instrument if fluorochromes not excited at 488 nm are being used
  • Additional reagents and equipment for cell culture and harvesting ( appendix 3B) and for immunophenotyping (unit 6.2)

Alternate Protocol 1: Determination of Cell Division Using Cell Trace Violet (CTV)

  • Cell Trace Violet (CTV; see recipe)
  • Multi‐laser flow cytometer with 405‐nm laser
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Literature Cited

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Internet Resources
  The Web site of Invitrogen, the supplier of CFSE/CFDA‐SE and CTV. This site contains much useful information on the application of the CFSE technique, and information on other alternative dyes, including CTV and the orange‐red emitting SNARF‐1 carboxylic acid, acetate, succinimidyl ester.
  The Web site of Verity Software House. ModFit software is used by many researchers to analyze proliferation data; it is available for both PC and Macintosh computers.
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