Chemistry:Carboxyfluorescein succinimidyl ester

From HandWiki
6-Carboxyfluorescein succinimidyl ester
Carboxyfluorescein succinimidyl ester.png
Names
Preferred IUPAC name
2,5-Dioxopyrrolidin-1-yl 3,6-dihydroxy-3-oxo-3H-spiro[[2]benzofuran-1,9′-xanthene]-6-carboxylate
Other names
CFSE; Carboxyfluorescein N-succinimidyl ester
Identifiers
3D model (JSmol)
ChemSpider
Properties
C25H15NO9
Molar mass 473.393 g·mol−1
Except where otherwise noted, data are given for materials in their standard state (at 25 °C [77 °F], 100 kPa).
☒N verify (what is ☑Y☒N ?)
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.

References

  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. ↑ "New fluorescent dyes for lymphocyte migration studies. Analysis by flow cytometry and fluorescence microscopy". Journal of Immunological Methods 133 (1): 87–97. October 1990. doi:10.1016/0022-1759(90)90322-M. PMID 2212694. 
  3. ↑ "Determination of lymphocyte division by flow cytometry". Journal of Immunological Methods 171 (1): 131–7. May 1994. doi:10.1016/0022-1759(94)90236-4. PMID 8176234. 
  4. ↑ "Carboxyfluorescein diacetate succinimidyl ester and the virgin lymphocyte: a marriage made in heaven". Immunology and Cell Biology 77 (6): 530–8. December 1999. doi:10.1046/j.1440-1711.1999.00871.x. PMID 10571674. 
  5. ↑ "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. July 1997. doi:10.1084/jem.186.2.239. PMID 9221753. 
  6. ↑ "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. August 1998. doi:10.1073/pnas.95.16.9488. PMID 9689107. Bibcode: 1998PNAS...95.9488G. 
  7. ↑ "Helper T cell differentiation is controlled by the cell cycle". Immunity 9 (2): 229–37. August 1998. doi:10.1016/S1074-7613(00)80605-6. PMID 9729043. 
  8. ↑ "B cell differentiation and isotype switching is related to division cycle number". The Journal of Experimental Medicine 184 (1): 277–81. July 1996. doi:10.1084/jem.184.1.277. PMID 8691143. 
  9. ↑ "Analysis of growth kinetics by division tracking". Immunology and Cell Biology 77 (6): 523–9. December 1999. doi:10.1046/j.1440-1711.1999.00869.x. PMID 10571673. 
  10. ↑ "A cellular calculus for signal integration by T cells". Nature Immunology 1 (3): 239–44. September 2000. doi:10.1038/79782. PMID 10973282. 
  11. ↑ "Estimating lymphocyte division and death rates from CFSE data". Bulletin of Mathematical Biology 68 (5): 1011–31. July 2006. doi:10.1007/s11538-006-9094-8. PMID 16832737. 
  12. ↑ "Modeling T- and B-cell growth and differentiation". Immunological Reviews 216: 119–29. April 2007. doi:10.1111/j.1600-065X.2006.00498.x. PMID 17367338. 
  13. ↑ "Measuring lymphocyte proliferation, survival and differentiation using CFSE time-series data". Nature Protocols 2 (9): 2057–67. 2007. doi:10.1038/nprot.2007.297. PMID 17853861. 
  14. ↑ "'Proliferative' and 'synthetic' airway smooth muscle cells are overlapping populations". Immunology and Cell Biology 82 (5): 471–8. October 2004. doi:10.1111/j.0818-9641.2004.01275.x. PMID 15479432. 
  15. ↑ "Insulin has a limited effect on the cell cycle progression in 3T3 L1 fibroblasts". Molecules and Cells 7 (6): 742–8. December 1997. PMID 9509415. 
  16. ↑ "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. February 2000. doi:10.1182/blood.V95.3.855.003k41_855_862. PMID 10648396. http://www.bloodjournal.org/cgi/pmidlookup?view=long&pmid=10648396. 
  17. ↑ "Flow cytometric analysis of Lactobacillus plantarum to monitor lag times, cell division and injury". Letters in Applied Microbiology 25 (4): 295–9. October 1997. doi:10.1046/j.1472-765x.1997.00225.x. PMID 9351280. 
  18. ↑ "Tumor-specific CD4+ T cells have a major "post-licensing" role in CTL mediated anti-tumor immunity". Journal of Immunology 165 (11): 6047–55. December 2000. doi:10.4049/jimmunol.165.11.6047. PMID 11086036. 
  19. ↑ "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. April 2004. doi:10.1182/blood-2003-06-2070. PMID 14630824. 
  20. ↑ "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. February 2004. doi:10.1016/j.jim.2003.10.017. PMID 14871532. 
  21. ↑ "An in vivo cytotoxicity threshold for influenza A virus-specific effector and memory CD8(+) T cells". Journal of Immunology 178 (3): 1285–92. February 2007. doi:10.4049/jimmunol.178.3.1285. PMID 17237374. 
  22. ↑ "Flow cytometric analysis of cell division by dye dilution". Current Protocols in Cytometry Chapter 9: Unit 9.11. February 2004. doi:10.1002/0471142956.cy0911s27. ISBN 0471142956. PMID 18770808. 
  23. ↑ "Monitoring lymphocyte proliferation in vitro and in vivo with the intracellular fluorescent dye carboxyfluorescein diacetate succinimidyl ester". Nature Protocols 2 (9): 2049–56. 2007. doi:10.1038/nprot.2007.296. PMID 17853860. 
  24. ↑ "Use of the intracellular fluorescent dye CFSE to monitor lymphocyte migration and proliferation". Current Protocols in Immunology Chapter 4: Unit4.9. February 2009. doi:10.1002/0471142735.im0409s84. ISBN 978-0471142737. PMID 19235770. 



Retrieved from "https://handwiki.org/wiki/index.php?title=Chemistry:Carboxyfluorescein_succinimidyl_ester&oldid=477815"