Carboxyfluorescein succinimidyl ester

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6-Carboxyfluorescein succinimidyl ester
Carboxyfluorescein succinimidyl ester.png
Other names
CFSE; Carboxyfluorescein N-succinimidyl ester
3D model (JSmol)
Molar mass 473.39 g·mol−1
Except where otherwise noted, data are given for materials in their standard state (at 25 °C [77 °F], 100 kPa).
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Infobox references

Carboxyfluorescein succinimidyl ester (CFSE) is a fluorescent cell staining dye. CFSE is cell permeable and covalently couples, via its succinimidyl group, to intracellular molecules,[1] notably, to intracellular lysine residues and other amine sources. Due to this covalent coupling reaction fluorescent CFSE can be retained within cells for extremely long periods. Also, due to this stable linkage, once incorporated within cells the dye is not transferred to adjacent cells.

CFSE is commonly confused with carboxyfluorescein diacetate succinimidyl ester (CFDA-SE), although they are not strictly the same molecule; CFDA-SE, due to its acetate groups, is highly cell permeable, while CFSE is much less so. As CFDA-SE, which is non-fluorescent, enters the cytoplasm of cells, intracellular esterases remove the acetate groups and convert the molecule to the fluorescent ester.

CFSE was originally developed as a fluorescent dye that could be used to stably label lymphocytes and track their migration within animals for many months.[2] Subsequent studies revealed that the dye can be used to monitor lymphocyte proliferation, both in vitro and in vivo, due to the progressive halving of CFSE fluorescence within daughter cells following each cell division.[3] The only limitation is that CFSE at high concentrations can be toxic for cells. However, when CFSE labelling is performed optimally, approximately 7-8 cell divisions can be identified before the CFSE fluorescence is too low to be distinguished above the autofluorescence background. Thus CFSE represents an extremely valuable fluorescent dye for immunological studies, allowing lymphocyte proliferation, migration and positioning to be simultaneously monitored. By the use of fluorescent antibodies against different lymphocyte cell surface markers it is also possible to follow the proliferation behaviour of different lymphocyte subsets.[4] In addition, unlike other methods, CFSE-labeled viable cells can be recovered for further analysis.

Since the initial description of CFSE it has been used in thousands of immunological studies, an example of an early proliferation study in animals being described by Kurts et al.[5] However, perhaps the most important CFSE investigations have been those demonstrating that many of the effector functions of lymphocytes, such as cytokine production by T lymphocytes,[6][7] and antibody class switching by B cells,[8] are division dependent. Sophisticated mathematical models have also been developed to analyse CFSE data and probe various aspects of immune responses.[9][10][11][12][13] Furthermore, the use of CFSE has extended beyond the immune system, with the dye being used to monitor the proliferation of many other cell types such as smooth muscle cells,[14] fibroblasts,[15] hematopoietic stem cells[16] and even bacteria.[17] Another novel application of CFSE is its use for the in vitro and in vivo determination of cytotoxic lymphocytes.[18][19][20][21]

Detailed protocols are now available that can be used to label lymphocytes (and other cell types) with a high degree of reliability and precision.[22][23][24] One of the most important parameters, however, is to ensure that the cell population being studied has not been too heavily labelled with CFSE as such cells, although remaining viable, proliferate sub-optimally.


  1. ^ Parish CR (December 1999). "Fluorescent dyes for lymphocyte migration and proliferation studies". Immunology and Cell Biology. 77 (6): 499–508. doi:10.1046/j.1440-1711.1999.00877.x. PMID 10571670.
  2. ^ Weston SA, Parish CR (October 1990). "New fluorescent dyes for lymphocyte migration studies. Analysis by flow cytometry and fluorescence microscopy". Journal of Immunological Methods. 133 (1): 87–97. doi:10.1016/0022-1759(90)90322-M. PMID 2212694.
  3. ^ Lyons AB, Parish CR (May 1994). "Determination of lymphocyte division by flow cytometry". Journal of Immunological Methods. 171 (1): 131–7. doi:10.1016/0022-1759(94)90236-4. PMID 8176234.
  4. ^ Fazekas de St Groth B, Smith AL, Koh WP, Girgis L, Cook MC, Bertolino P (December 1999). "Carboxyfluorescein diacetate succinimidyl ester and the virgin lymphocyte: a marriage made in heaven". Immunology and Cell Biology. 77 (6): 530–8. doi:10.1046/j.1440-1711.1999.00871.x. PMID 10571674.
  5. ^ Kurts C, Kosaka H, Carbone FR, Miller JF, Heath WR (July 1997). "Class I-restricted cross-presentation of exogenous self-antigens leads to deletion of autoreactive CD8(+) T cells". The Journal of Experimental Medicine. 186 (2): 239–45. doi:10.1084/jem.186.2.239. PMC 2198972. PMID 9221753.
  6. ^ Gett AV, Hodgkin PD (August 1998). "Cell division regulates the T cell cytokine repertoire, revealing a mechanism underlying immune class regulation". Proceedings of the National Academy of Sciences of the United States of America. 95 (16): 9488–93. doi:10.1073/pnas.95.16.9488. PMC 21365. PMID 9689107.
  7. ^ Bird JJ, Brown DR, Mullen AC, et al. (August 1998). "Helper T cell differentiation is controlled by the cell cycle". Immunity. 9 (2): 229–37. doi:10.1016/S1074-7613(00)80605-6. PMID 9729043.
  8. ^ Hodgkin PD, Lee JH, Lyons AB (July 1996). "B cell differentiation and isotype switching is related to division cycle number". The Journal of Experimental Medicine. 184 (1): 277–81. doi:10.1084/jem.184.1.277. PMC 2192686. PMID 8691143.
  9. ^ Nordon RE, Nakamura M, Ramirez C, Odell R (December 1999). "Analysis of growth kinetics by division tracking". Immunology and Cell Biology. 77 (6): 523–9. doi:10.1046/j.1440-1711.1999.00869.x. PMID 10571673.
  10. ^ Gett AV, Hodgkin PD (September 2000). "A cellular calculus for signal integration by T cells". Nature Immunology. 1 (3): 239–44. doi:10.1038/79782. PMID 10973282.
  11. ^ De Boer RJ, Ganusov VV, Milutinović D, Hodgkin PD, Perelson AS (July 2006). "Estimating lymphocyte division and death rates from CFSE data". Bulletin of Mathematical Biology. 68 (5): 1011–31. doi:10.1007/s11538-006-9094-8. PMID 16832737.
  12. ^ Callard R, Hodgkin P (April 2007). "Modeling T- and B-cell growth and differentiation". Immunological Reviews. 216: 119–29. doi:10.1111/j.1600-065X.2006.00498.x. PMID 17367338.
  13. ^ Hawkins ED, Hommel M, Turner ML, Battye FL, Markham JF, Hodgkin PD (2007). "Measuring lymphocyte proliferation, survival and differentiation using CFSE time-series data". Nature Protocols. 2 (9): 2057–67. doi:10.1038/nprot.2007.297. PMID 17853861.
  14. ^ Sukkar MB, Stanley AJ, Blake AE, et al. (October 2004). "'Proliferative' and 'synthetic' airway smooth muscle cells are overlapping populations". Immunology and Cell Biology. 82 (5): 471–8. doi:10.1111/j.0818-9641.2004.01275.x. PMID 15479432.
  15. ^ Khil LY, Kim JY, Yoon JB, et al. (December 1997). "Insulin has a limited effect on the cell cycle progression in 3T3 L1 fibroblasts". Molecules and Cells. 7 (6): 742–8. PMID 9509415.
  16. ^ Oostendorp RA, Audet J, Eaves CJ (February 2000). "High-resolution tracking of cell division suggests similar cell cycle kinetics of hematopoietic stem cells stimulated in vitro and in vivo". Blood. 95 (3): 855–62. PMID 10648396.
  17. ^ Ueckert JE, Nebe von-Caron G, Bos AP, ter Steeg PF (October 1997). "Flow cytometric analysis of Lactobacillus plantarum to monitor lag times, cell division and injury". Letters in Applied Microbiology. 25 (4): 295–9. doi:10.1046/j.1472-765x.1997.00225.x. PMID 9351280.
  18. ^ Marzo AL, Kinnear BF, Lake RA, et al. (December 2000). "Tumor-specific CD4+ T cells have a major "post-licensing" role in CTL mediated anti-tumor immunity". Journal of Immunology. 165 (11): 6047–55. doi:10.4049/jimmunol.165.11.6047. PMID 11086036.
  19. ^ Jedema I, van der Werff NM, Barge RM, Willemze R, Falkenburg JH (April 2004). "New CFSE-based assay to determine susceptibility to lysis by cytotoxic T cells of leukemic precursor cells within a heterogeneous target cell population". Blood. 103 (7): 2677–82. doi:10.1182/blood-2003-06-2070. PMID 14630824.
  20. ^ Hermans IF, Silk JD, Yang J, et al. (February 2004). "The VITAL assay: a versatile fluorometric technique for assessing CTL- and NKT-mediated cytotoxicity against multiple targets in vitro and in vivo". Journal of Immunological Methods. 285 (1): 25–40. doi:10.1016/j.jim.2003.10.017. PMID 14871532.
  21. ^ Stambas J, Doherty PC, Turner SJ (February 2007). "An in vivo cytotoxicity threshold for influenza A virus-specific effector and memory CD8(+) T cells". Journal of Immunology. 178 (3): 1285–92. doi:10.4049/jimmunol.178.3.1285. PMID 17237374.
  22. ^ Lyons AB, Doherty KV (February 2004). "Flow cytometric analysis of cell division by dye dilution". Current Protocols in Cytometry. Chapter 9: Unit 9.11. doi:10.1002/0471142956.cy0911s27. PMID 18770808.
  23. ^ Quah BJ, Warren HS, Parish CR (2007). "Monitoring lymphocyte proliferation in vitro and in vivo with the intracellular fluorescent dye carboxyfluorescein diacetate succinimidyl ester". Nature Protocols. 2 (9): 2049–56. doi:10.1038/nprot.2007.296. PMID 17853860.
  24. ^ Parish CR, Glidden MH, Quah BJ, Warren HS (February 2009). "Use of the intracellular fluorescent dye CFSE to monitor lymphocyte migration and proliferation". Current Protocols in Immunology. Chapter 4: Unit4.9. doi:10.1002/0471142735.im0409s84. PMID 19235770.