Superior properties of CellTrace Yellow™ as a division tracking dye for human and murine lymphocytes.

Jessica C Tempany,Vanessa L Bryant,Jie HS Zhou,Philip D Hodgkin

doi:10.1111/imcb.1020

Abstract

The discovery of cell division tracking properties of 5‐(and‐6)‐carboxyfluorescein diacetate succinimidyl ester (CFSE) by Lyons and Parish in 1994 led to a broad range of new methods and numerous important biological discoveries. After labeling, CFSE is attached to free amine groups and intracellular proteins in the cytoplasm and nucleus of a cell, and halves in fluorescence intensity with each round of cell division, enabling enumeration of the number of divisions a cell has undergone. A range of popular division tracking dyes were subsequently developed, including CellTrace Violet (CTV), making available the green fluorescent channel previously occupied by CFSE. More recently, CellTrace Yellow (CTY) and CellTrace Far Red (CTFR), each with unique fluorescence properties, were introduced. In a comparison, we found that the fluorescence values of both dyes were well separated from autofluorescence, and enabled a greater number of divisions to be identified than CTV, before this limit was reached. These new dyes provided clear and well‐separated peaks for both murine and human B lymphocytes, and should find wide application. The range of excitation/emission spectra available for division tracking dyes now also facilitates multiplexing, that is, the labeling of cells with different combinations of dyes to give a unique fluorescence signature, allowing single cell in vitro and in vivo tracking. The combinatorial possibilities are significantly increased with these additional dyes.

Highlights

The immune system is a complex and carefully-regulated environment where lymphocytes coordinate their proliferation, differentiation, survival and migration behavior to orchestrate an appropriate response
In practice, application of this principle is limited by the toxicity of the dye and/or the final level of dimethyl sulfoxide (DMSO) solvent used with each dye
CTY 20 μmol L–1 Unstimulated Unlabeled this report, we have outlined a number of significant advantages of utilizing new generation cell tracking dyes, and in particular, CellTrace Yellow (CTY)

Summary

Introduction

The immune system is a complex and carefully-regulated environment where lymphocytes coordinate their proliferation, differentiation, survival and migration behavior to orchestrate an appropriate response. The use of fluorescent dye-labeling to interrogate ongoing lymphocyte responses, both in vitro and in vivo has led to significant advances in our understanding of immune regulation.[1,2,3] The most widely used division tracking dye method to date, labeling cells with 5-(and-6)carboxyfluorescein diacetate succinimidyl ester (CFSE), was introduced in 1994 by Lyons and Parish.[1] This dye incorporates into both the cytoplasm and nucleus of cells, and is well-retained within stained cells.[4] Upon division, CFSE distributes evenly between daughter cells, resulting in a twofold dilution with each consecutive cell division, forming distinct peaks when a proliferating population is viewed as a histogram following flow cytometry analysis.[1] This method enables determination of the number of divisions each cell has undergone, until the CFSE fluorescence is too dilute to be distinguishable from autofluorescence of the cell. The reduced intensity of labeling[5,6] and poor definition of division peaks of these dyes have limited their applications.[7]

Methods

Results

Conclusion