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Advanced Application of CFSE for Cellular Tracking

Jacek M. Witkowski¹

¹Medical University of Gdańsk, Gdańsk, Poland

Publication Name:

Current Protocols in Cytometry

Unit Number:

Unit 9.25

DOI:

10.1002/0471142956.cy0925s44

Online Posting Date:

April, 2008

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Abstract

This unit proposes a method to extend the already well known dividing-cell-tracking (DCT) cytometric technique based on supravital staining of the lymphocytes with CFSE and allowing them to divide afterwards, beyond simple observation and counting of dividing cells and their generations. Dynamic proliferation parameters that make it possible to determine for in vitro dividing human lymphocytes from various sources, are the actual duration of the pre-division transition period (G0G1), time of a single division, and number of divisions an average dividing cell performs over the time of an experiment, as well as the number of effective precursors giving rise to viable daughter lymphocytes. As the method does not require purification of the lymphocyte population of interest, yet allows the calculations for any cytometrically discernible subpopulation, it presents a powerful tool for detailed analysis of the efficiency of proliferative response of the immune cells. Curr. Protocol. Cytom. 44:9.25.1-9.25.8. © 2008 by John Wiley & Sons, Inc.

Keywords: dividing cell tracking; CFSE; human lymphocytes; cell cycle length; number of divisions; transition time; effective precursors

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Introduction
Strategic Planning
Basic Protocol: Determination of Numerical Values of Lymphocyte Division Parameters
Reagents and Solutions
Commentary
Literature Cited
Figures

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Materials

Basic Protocol: Determination of Numerical Values of Lymphocyte Division Parameters

Materials

5 mM 5-(and 6-) carboxyfluorescein diacetate succinimidyl ester (CFSE, see recipe)
Single-cell suspension of cells of interest (see above; prepare suspensions, e.g., as in Coligan et al., 2008, Chapter 7)
RPMI 1640/0.5% (w/v) BSA or RPMI 1640/0.5% (v/v) FBS
Complete RPMI with 10% FBS (appendix 2A)
PE-conjugated Annexin V solution (alternatively, Hoechst 33342 solution) for estimation of proportion of apoptotic cells in consecutive generations of dividing lymphocytes (also see unit 7.19)
Antibodies for immunophenotyping tagged with PE, PerCP, or fluorochromes emitting light of longer wavelength when excited with 488-nm argon laser
Flow cytometer with 488-nm argon laser and at least three fluorescence-acquisition channels; optionally, a multi-laser instrument with UV excitation and blue light emission detection, or a multi-laser, multicolor instrument

Additional reagents and equipment for cell culture and harvesting (appendix 3B), staining for apoptosis (unit 7.19), immunophenotyping (units 6.2 & 6.5), staining for cell viability (unit 9.2), and determination of absolute cell number (unit 6.8)

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Figures

Figure 9.25.1

Representative results for human PBMC stimulation and CFSE division tracking of CD4⁺ lymphocytes. Flow data presentation as density plot allows for easy distinction of consecutive generations of dividing cells. Arrows indicate the region containing generation 2 in the plot of the sample cultured for 72 hr (A) and 120 hr (B). Actual numbers of cells in the generations are shown.

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Literature Cited

Literature Cited
	Coligan, J.E., Bierer, B.E., Margulies, D.H., Shevach, E.M., and Strober, W. (eds.) 2008. Current Protocols in Immunology. John Wiley & Sons, Hoboken, N.J.
	Deenick, E.K., Gett, A.V., and Hodgkin, P.D. 2003. Stochastic model of T cell proliferation: A calculus revealing IL-2 regulation of precursor frequencies, cell cycle time, and survival. J. Immunol. 170: 4963-4972.
	Lyons, A.B. and Parish, C.R. 1994. Determination of lymphocyte division by flow cytometry. J. Immunol. Meth. 171: 131-137.
	Mintern, J., Li, M., Davey, G.M., Blanas, E., Kurts, C., Carbone, F.R., and Heath, W.R. 1999. The use of carboxyfluorescein diacetate succinimidyl ester to determine the site, duration and cell type responsible for antigen presentation in vivo. Immunol. Cell Biol. 77: 539-543.
	Nordon, R.E., Nakamura, M., Ramirez, C., and Odell, R. 1999. Analysis of growth kinetics by division tracking. Immunol. Cell Biol. 77: 523-529.
	Witkowski, J.M. and Bryl, E. 2004. Paradoxical age-related cell cycle quickening of human CD4+ lymphocytes: A role for cyclin D1 and calpain. Exp. Gerontol. 39: 577-558.