Monitoring lymphocyte proliferation in vitro and in vivo with the intracellular fluorescent dye carboxyfluorescein diacetate succinimidyl ester

Nat Protoc. 2007;2(9):2049-56. doi: 10.1038/nprot.2007.296.


This protocol outlines the carboxyfluorescein diacetate succinimidyl ester (CFSE) method for following the proliferation of human lymphocytes in vitro and mouse lymphocytes both in vitro and in vivo. The method relies on the ability of CFSE to covalently label long-lived intracellular molecules with the highly fluorescent dye, carboxyfluorescein. Following each cell division, the equal distribution of these fluorescent molecules to progeny cells results in a halving of the fluorescence of daughter cells. The CFSE labeling protocol described, which typically takes <1 h to perform, allows the detection of up to eight cell divisions before CFSE fluorescence is decreased to the background fluorescence of unlabeled cells. Protocols are outlined for labeling large and small numbers of human and mouse lymphocytes, labeling conditions being identified that minimize CFSE toxicity but maximize the number of cell divisions detected. An important feature of the technique is that division-dependent changes in the expression of cell-surface markers and intracellular proteins are easily quantified by flow cytometry.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • CD4-Positive T-Lymphocytes / cytology
  • CD4-Positive T-Lymphocytes / immunology*
  • Cell Proliferation
  • Cells, Cultured
  • Flow Cytometry / methods*
  • Fluoresceins*
  • Fluorescent Dyes*
  • Humans
  • Lymphocyte Activation*
  • Mice
  • Staining and Labeling
  • Succinimides*


  • 5-(6)-carboxyfluorescein diacetate succinimidyl ester
  • Fluoresceins
  • Fluorescent Dyes
  • Succinimides