Tracking keratinocytes and melanocytes using carboxyfluorescein hydroxysuccinimidyl ester staining

PLoS One. 2019 Aug 29;14(8):e0221878. doi: 10.1371/journal.pone.0221878. eCollection 2019.


Introduction: The treatment of burn wounds and hypopigmentation conditions often require autologous transplantation of keratinocytes and melanocytes. Tracking transplanted cells to ascertain their contribution to tissue recapitulation presents a challenge. This study demonstrates a methodology based on passive staining with carboxyfluorescein hydroxysuccinimidyl ester (CFSE) that enables localization of cells in tissue sections to investigate the fate of transplanted cells in wound re-epithelialisation.

Methods: Viability and migration of CFSE-stained keratinocytes and melanocytes were investigated using viability staining and scratch assays, while proliferation of cells was measured using flow cytometry. In addition, CFSE-stained keratinocytes and melanocytes were transplanted to a human ex vivo wound model, either in suspension, or with the aid of macroporous gelatine microcarriers. Wounds were analysed seven, 14 and 21 days post transplantation using cryosectioning and fluorescence microscopy. Sections from wounds with transplanted co-cultured keratinocytes and melanocytes were stained for pancytokeratin to distinguish keratinocytes.

Results: CFSE-staining of keratinocytes and melanocytes did not affect the viability, migration or proliferation of the cells. Transplanted cells were tracked in ex vivo wounds for 21 days, illustrating that the staining had no effect on wound re-epithelialisation. In conclusion, this study presents a novel application of CFSE-staining for tacking transplanted primary human keratinocytes and melanocytes.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cell Survival
  • Fluoresceins*
  • Fluorescent Antibody Technique* / methods
  • Fluorescent Dyes*
  • Humans
  • Immunohistochemistry
  • Keratinocytes / metabolism*
  • Melanocytes / metabolism*
  • Microscopy, Fluorescence
  • Succinimides*


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

Grants and funding

This study was funded by the Region Östergötland (grant number LIO-695901 awarded to Professor Gunnar Kratz). The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.