Carboxyfluorescein succinimidyl ester

Last updated
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
PubChem CID
  • InChI=1S/C25H15NO9/c27-13-2-5-16-19(10-13)33-20-11-14(28)3-6-17(20)25(16)18-9-12(1-4-15(18)24(32)34-25)23(31)35-26-21(29)7-8-22(26)30/h1-6,9-11,27-28H,7-8H2 X mark.svgN
    Key: VDABVNMGKGUPEY-UHFFFAOYSA-N X mark.svgN
  • InChI=1/C25H15NO9/c27-13-2-5-16-19(10-13)33-20-11-14(28)3-6-17(20)25(16)18-9-12(1-4-15(18)24(32)34-25)23(31)35-26-21(29)7-8-22(26)30/h1-6,9-11,27-28H,7-8H2
    Key: VDABVNMGKGUPEY-UHFFFAOYAV
  • O=C(C5=C4C=C(C(ON6C(CCC6=O)=O)=O)C=C5)OC24C3=C(C=C(O)C=C3)OC1=CC(O)=CC=C12
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).
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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.

Related Research Articles

Immunology Branch of medicine studying the immune system

Immunology is a branch of biology that covers the study of immune systems in all organisms. Immunology charts, measures, and contextualizes the physiological functioning of the immune system in states of both health and diseases; malfunctions of the immune system in immunological disorders ; and the physical, chemical, and physiological characteristics of the components of the immune system in vitro, in situ, and in vivo. Immunology has applications in numerous disciplines of medicine, particularly in the fields of organ transplantation, oncology, rheumatology, virology, bacteriology, parasitology, psychiatry, and dermatology.

Natural killer cell Type of cytotoxic lymphocyte

Natural killer cells, also known as NK cells or large granular lymphocytes (LGL), are a type of cytotoxic lymphocyte critical to the innate immune system that belong to the rapidly expanding family of innate lymphoid cells (ILC) and represent 5–20% of all circulating lymphocytes in humans. The role of NK cells is analogous to that of cytotoxic T cells in the vertebrate adaptive immune response. NK cells provide rapid responses to virus-infected cell and other intracellular pathogens acting at around 3 days after infection, and respond to tumor formation. Typically, immune cells detect the major histocompatibility complex (MHC) presented on infected cell surfaces, triggering cytokine release, causing the death of the infected cell by lysis or apoptosis. NK cells are unique, however, as they have the ability to recognize and kill stressed cells in the absence of antibodies and MHC, allowing for a much faster immune reaction. They were named "natural killers" because of the notion that they do not require activation to kill cells that are missing "self" markers of MHC class 1. This role is especially important because harmful cells that are missing MHC I markers cannot be detected and destroyed by other immune cells, such as T lymphocyte cells.

Immunotherapy or biological therapy is the treatment of disease by activating or suppressing the immune system. Immunotherapies designed to elicit or amplify an immune response are classified as activation immunotherapies, while immunotherapies that reduce or suppress are classified as suppression immunotherapies.

Cytotoxicity is the quality of being toxic to cells. Examples of toxic agents are an immune cell or some types of venom, e.g. from the puff adder or brown recluse spider.

Flow cytometry Lab technique in biology and chemistry

Flow cytometry (FC) is a technique used to detect and measure physical and chemical characteristics of a population of cells or particles.

Fluorescein Chemical compound

Fluorescein is an organic compound and dye. It is available as a dark orange/red powder slightly soluble in water and alcohol. It is widely used as a fluorescent tracer for many applications.

Granzymes are serine proteases released by cytoplasmic granules within cytotoxic T cells and natural killer (NK) cells. They induce programmed cell death (apoptosis) in the target cell, thus eliminating cells that have become cancerous or are infected with viruses or bacteria. Granzymes also kill bacteria and inhibit viral replication. In NK cells and T cells, granzymes are packaged in cytotoxic granules along with perforin. Granzymes can also be detected in the rough endoplasmic reticulum, golgi complex, and the trans-golgi reticulum. The contents of the cytotoxic granules function to permit entry of the granzymes into the target cell cytosol. The granules are released into an immune synapse formed with a target cell, where perforin mediates the delivery of the granzymes into endosomes in the target cell, and finally into the target cell cytosol. Granzymes are part of the serine esterase family. They are closely related to other immune serine proteases expressed by innate immune cells, such as neutrophil elastase and cathepsin G.

Antibody-dependent cellular cytotoxicity Cell-mediated killing of other cells mediated by antibodies

Antibody-dependent cellular cytotoxicity (ADCC), also referred to as antibody-dependent cell-mediated cytotoxicity, is a mechanism of cell-mediated immune defense whereby an effector cell of the immune system actively lyses a target cell, whose membrane-surface antigens have been bound by specific antibodies. It is one of the mechanisms through which antibodies, as part of the humoral immune response, can act to limit and contain infection.

Immunological synapse Interface between lymphocyte and target cell

In immunology, an immunological synapse is the interface between an antigen-presenting cell or target cell and a lymphocyte such as a T/B cell or Natural Killer cell. The interface was originally named after the neuronal synapse, with which it shares the main structural pattern. An immunological synapse consists of molecules involved in T cell activation, which compose typical patterns—activation clusters. Immunological synapses are the subject of much ongoing research.

6-Carboxyfluorescein Chemical compound

6-Carboxyfluorescein (6-FAM) is a fluorescent dye with an absorption wavelength of 495 nm and an emission wavelength of 517 nm. A carboxyfluorescein molecule is a fluorescein molecule with a carboxyl group added. They are commonly used as a tracer agents. It is used in the sequencing of nucleic acids and in the labeling of nucleotides.

Antigen presentation Vital immune process that is essential for T cell immune response triggering

Antigen presentation is a vital immune process that is essential for T cell immune response triggering. Because T cells recognize only fragmented antigens displayed on cell surfaces, antigen processing must occur before the antigen fragment, now bound to the major histocompatibility complex (MHC), is transported to the surface of the cell, a process known as presentation, where it can be recognized by a T-cell receptor. If there has been an infection with viruses or bacteria, the cell will present an endogenous or exogenous peptide fragment derived from the antigen bound to MHC molecules. There are two types of MHC molecules which differ in the behaviour of the antigens: MHC class I molecules (MHC-I) bind peptides from the cell cytosol, while peptides generated in the endocytic vesicles after internalisation are bound to MHC class II (MHC-II). Cellular membranes separate these two cellular environments - intracellular and extracellular. Each T cell can only recognize tens to hundreds of copies of a unique sequence of a single peptide among thousands of other peptides presented on the same cell, because an MHC molecule in one cell can bind to quite a large range of peptides.

Carboxyfluorescein diacetate succinimidyl ester Chemical compound

Carboxyfluorescein diacetate succinimidyl ester (CFDA-SE) is a cell permeable dye generally used in animal cell proliferation research. High concentrations of the dye are toxic to animal cells, however concentrations in the region of 10 micromolar are typically sufficient to give strong staining with minimal cell death.

Macrophage-1 antigen is a complement receptor ("CR3") consisting of CD11b and CD18.

Cancer immunology

Cancer immunology is an interdisciplinary branch of biology that is concerned with understanding the role of the immune system in the progression and development of cancer; the most well known application is cancer immunotherapy, which utilises the immune system as a treatment for cancer. Cancer immunosurveillance and immunoediting are based on protection against development of tumors in animal systems and (ii) identification of targets for immune recognition of human cancer.

HAVCR2

Hepatitis A virus cellular receptor 2 (HAVCR2), also known as T-cell immunoglobulin and mucin-domain containing-3 (TIM-3), is a protein that in humans is encoded by the HAVCR2 (TIM-3)gene. HAVCR2 was first described in 2002 as a cell surface molecule expressed on IFNγ producing CD4+ Th1 and CD8+ Tc1 cells. Later, the expression was detected in Th17 cells, regulatory T-cells, and innate immune cells. HAVCR2 receptor is a regulator of the immune response.

Trogocytosis

Trogocytosis is a process whereby lymphocytes conjugated to antigen-presenting cells extract surface molecules from these cells and express them on their own surface. The molecular reorganization occurring at the interface between the lymphocyte and the antigen-presenting cell during conjugation is also called "immunological synapse".

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PD-1 and PD-L1 inhibitors

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Complement-dependent cytotoxicity (CDC) is an effector function of IgG and IgM antibodies. When they are bound to surface antigen on target cell, the classical complement pathway is triggered by bonding protein C1q to these antibodies, resulting in formation of a membrane attack complex (MAC) and target cell lysis.

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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. ↑ 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. S2CID   5696309.
  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. hdl: 1874/22578 . 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. doi:10.1182/blood.V95.3.855.003k41_855_862. 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. S2CID   1984056.
  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. ISBN   0471142956. 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. ISBN   978-0471142737. PMID   19235770.
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