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 JoVE Immunology and Infection

The Use of Carboxyfluorescein Diacetate Succinimidyl Ester (CFSE) to Monitor Lymphocyte Proliferation

1, 1

1Department of Immunology, John Curtin School of Medical Research, Australian National University

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    Summary

    CFSE covalently labels long-lived intracellular molecules with the fluorescent dye, carboxyfluorescein. As such, when a CFSE-labeled cell divides, its progeny have half the amount of fluorescence, which can thereby be used to assess cell division. This article describes the procedures typically used for labeling mouse lymphocytes with CFSE.

    Date Published: 10/12/2010, Issue 44; doi: 10.3791/2259

    Cite this Article

    Quah, B. J. C., Parish, C. R. The Use of Carboxyfluorescein Diacetate Succinimidyl Ester (CFSE) to Monitor Lymphocyte Proliferation. J. Vis. Exp. (44), e2259, doi:10.3791/2259 (2010).

    Abstract

    Carboxyfluorescein succinimidyl ester (CFSE) is an effective and popular means to monitor lymphocyte division1-3. CFSE covalently labels long-lived intracellular molecules with the fluorescent dye, carboxyfluorescein. Thus, when a CFSE-labeled cell divides, its progeny are endowed with half the number of carboxyfluorescein-tagged molecules and thus each cell division can be assessed by measuring the corresponding decrease in cell fluorescence via Flow cytometry. The capacity of CFSE to label lymphocyte populations with a high fluorescent intensity of exceptionally low variance, coupled with its low cell toxicity, make it an ideal dye to measure cell division. Since it is a fluorescein-based dye it is also compatible with a broad range of other fluorochromes making it applicable to multi-color flow cytometry. This article describes the procedures typically used for labeling mouse lymphocytes for the purpose of monitoring up to 8 cell divisions. These labeled cells can be used both for in vitro and in vivo studies.

    Protocol

    1) Reagent Setup

    1.1) CFSE stock solution

    The CFSE dye is purchased as carboxyfluorescein diacetate succinimidyl ester (CFDA, SE) (MW 557.47 g/mole), typically in 25 mg vials. Dissolve the 25 mg in 8.96 mL DMSO for a final stock solution of 5 mM (store as 50-100 μL aliquots at -20 °C for several months). CFDA, SE will react with aqueous solution so it is critical that such exposure be avoided during storage.

    1.2) Lymphocytes

    Isolate lymphocytes from spleen and or lymph nodes from mice and resuspend in 1 mL of culture medium (e.g. RPMI) with 5% heat-inactivated (HI) fetal calf serum (FCS), with a cell concentration of between 0.5 x 106 - 10 x 107/mL.

    2) CFSE Labeling

    2.1) Routine method:

    1. Thoroughly resuspend cells in the 1 mL volume of medium and place carefully in the bottom of a fresh (non-wetted) 10 mL conical tube.
    2. Lay the tube horizontally (using a non-wetted tube will prevent the 1 mL-cell suspension from moving and prematurely mixing with the CFDA, SE solutions).
    3. Carefully add 110 μL of PBS to the non-wetted portion of the plastic at the top of the tube ensuring it does not make contact with the cell solution.
    4. Resuspend 1.1 μL of the 5 mM stock of CFDA, SE in the 110 μL PBS.
    5. Quickly cap the tube and invert and vortex well to get quick uniform mixing of the solutions Note that the CFSE dye is at a final concentration of 5 μM, however, the optimum concentration may have to be determined for each batch prepared, and then checked every 6 months to ensure it is effective.
    6. Incubate cells for 5 min at room temperature. Note that upon conversion of CFDA, SE to CFSE (see discussion) the dye becomes fluorescent and thus prone to bleaching by exposure to excessive light. Thus, it is advisable to protect the tube from light from this point on, for example by covering it with aluminum foil.
    7. Wash cells by diluting in 10 volumes of 20 °C PBS containing 5 % HI FCS, sedimenting by centrifugation at 300 x g for 5 min at 20 °C and discarding the supernatant Repeat wash twice more.

    2.2) Alternative method, which is also appropriate for higher cell numbers (10 - 30 x 107):

    1. Prepare a 2 x (10 μM) CFDA, SE solution by adding 2 μL of the 5 mM stock to 1 mL of PBS and quickly add this to 1 mL of thoroughly resuspended cells, then quickly cap the tube and invert and vortex well to get quick uniform mixing.
    2. Incubate the cells for 5 min at room temperature and wash as in steps 2.1(f) and 2.1(g).

    2.3) Cells can then be applied to a protocol of interest for induction of cell division. Labeled cells can be used in both in vitro and in vivo assays.

    2.4) At the completion of the proliferation assay, cells are harvested and analyzed by flow cytometry (see below for representative examples).

    3) Representative Results

    To assess lymphocyte proliferation by CFSE dilution, it is useful to know the fluorescence of undivided cells (i.e. maximum fluorescence) and non-labeled cells (i.e. minimum fluorescence). Therefore, your experimental design should take these controls into account. Below are examples of in vitro and in vivo assays using CFSE to measure CD8+ T cell division by flow cytometry.

    3.1) In vitro stimulation

    Figure 1 shows the CFSE profiles of CFSE-labeled ovalbumin (OVA)-specific T cell receptor transgenic CD8+ OT-I T cells after 3 days culture with dendritic cells pulsed with various amounts of OVA. CD8+ T cells purified from the spleen and lymph nodes of OT-I mice were labeled with CFSE and 1 x 105 cells cultured with 3.3 x 104 DC. DC where pulsed with OVA for 1 hr at 37 °C and washed twice prior to culture. After 3 days, cells where harvested and labeled with anti-CD8-APC antibodies and Hoechst 33258 and then analyzed by flow cytometry (50,000 events collected). Live (Hoechst 33258-) CD8+ cells are shown. As controls, CD8+ T cells cultured alone, shows the CFSE intensity of non-divided cells, while the non-labeled cells shows the auto-fluorescence of the cells and the limits of detectable cell divisions. Note that 4 CFSE peaks can be seen in each of the panels in this example, indicating that the cells have undergone up to 3 divisions.

    3.2) In vivo stimulation

    Figure 2 shows the CFSE profiles of CFSE-labeled OVA-specific T cell receptor transgenic CD8+ OT-I T cells after 3 days in vivo in the presence of 20 μg OVA. CD8+ T cells purified from the spleen and lymph nodes of CD45.1 congenic OT-I mice were labeled with CFSE and 5 x 106 cells injected i.v. into the lateral tail vein of C56BL/6J (CD45.2+) mice. After 2 hr mice were injected i.v. with 20 μg of OVA. After 3 days, spleens from the mice were collected, made into a single cell suspension, labeled with anti-B220-PerCP, anti-CD8-APC-Cy7 and anti-CD45.1-PE-Cy7 antibodies and Hoechst 33258 and then analyzed by flow cytometry (1,000,000 events collected). Live (Hoechst 33258-) B220- cells are shown in left panel and gated CD8+ CD45.1+ cells shown in right panel. As controls, unstimulated CFSE-labeled CD8+ T cells, shows the CFSE intensity of non-divided cells, while the non-labeled cells shows the auto-fluorescence of the cells and the limits of detectable cell divisions. Note that the use of the CD45 allotypic difference is vital in resolving the transferred CD8+ T cells from host, as they are a minor subset of the total CD8+ T cells (left panel). Note that most cells fall within 7 CFSE peaks in this example (right panel), indicating that the cells have undergone up to 6 divisions.

    Figure 1
    Figure 1. Ability of CFSE-labeled ovalbumin (OVA)-specific T cell receptor transgenic CD8+ OT-I T cells to respond in vitro to DC pulsed with different concentrations of OVA, data shown being after 3 days culture. Note non-divided population of CD8+ T cells in the absence of antigen (light grey histogram) and auto-fluorescence of non-labeled population (dark grey histogram). The Figure depicts CD8+ T cells that have divided 1-3 times based on CFSE dilution peaks, with more T cells dividing at the higher antigen concentrations.

    Figure 2
    Figure 2. Ability of CFSE-labeled ovalbumin (OVA)-specific T cell receptor transgenic CD8+ OT-I T cells, when adoptively transferred into C57BL/6 mice to respond to OVA, data shown 3 days after adoptive transfer. Left panel: CD8+, CD45.1+ OT-1 T cells in recipient mice. Right panel: CFSE proliferation profile. Note non-divided population of CD8+ T cells in recipient mice not receiving antigen (light grey histogram) and auto-fluorescence of non-labeled lymphocytes (dark grey histogram). The Figure depicts CD8+ T cells that have divided 1-6 times based on CFSE dilution peaks.

    Figure 3
    Figure 3. A representation of the various molecular events that occur during the labeling of cells with carboxyfluorescein diacetate succinimidyl ester (CFDA, SE). For details of the process see Discussion text. Note: The generic acronym 'CFSE', not CFDA, SE, is used to describe the labeling reagent and the labeling procedure in general. Figure based on Parish4.

    Discussion

    In order to appreciate how CFSE works and why rapid uniform labeling is required for its use in proliferation analysis, it is useful to understand the molecular basis of CFSE cell labeling. This can be divided into two phases, 1) Cell entry and 2) Protein labeling (Figure 3). 1) Cell entry: Entry of the dye in to the cell is mediated by the diacetylated form of the dye, carboxyfluorescein diacetate succinimidyl ester (CFDA, SE). The acetates make the dye highly membrane permeant allowing its rapid flux across the plasma membrane. Esterases, present within the cell, cleave the acetates from CFDA, SE and thereby give rise to the CFSE form of the dye which is much less membrane permeant, thus concentrating the dye within the cell. 2) Protein labeling: While both CFDA, SE and CFSE have amino-reactive succinimidyl side chains, only the CFSE is fluorescent. The high intracellular concentration of CFSE facilitates rapid and high level intracellular labeling of proteins. CFSE labeling of cells must be performed quickly in order to obtain a homogenously labeled population of cells, which is critical for resolving cells that have undergone several divisions as shown in the representative results (Figure 1 and 2).

    Disclosures

    No conflicts of interest declared.

    Acknowledgements

    The work reported in this article was supported by a National Health and Medical Research Council (NHMRC) of Australia Program Grant (CRP) and a NHMRC Project Grant (CRP and BJCQ). Also BJCQ is a NHMRC Peter Doherty Postdoctoral Fellow.

    Materials

    Name Company Catalog Number Comments
    CFDA,SE Molecular Probes, Life Technologies
    DMSO Cambridge Isotope Laboratories
    Lymphocytes eg mouse or human
    RPMI GIBCO, by Life Technologies
    FCS SAFC Global
    10-15ml conical tubes Falcon BD
    PBS GIBCO, by Life Technologies
    Aluminium foil Capral Aluminium
    Vortex Scientific Industries Inc.
    Centrifuge Eppendorf
    Flow cytometer BD Biosciences

    References

    1. Lyons, A. B. & Parish, C. R. Determination of lymphocyte division by flow cytometry. J. Immunol. Methods 171, 131-137 (1994).
    2. Parish, C. R., Glidden, M. H., Quah, B. J., & Warren, H. S. Use of the intracellular fluorescent dye CFSE to monitor lymphocyte migration and proliferation. Current protocols in immunology / edited by John E. Coligan ... [et al. Chapter 4, Unit 4 9 (2009).
    3. Quah, B. J., Warren, H. S., & Parish, C. R. Monitoring lymphocyte proliferation in vitro and in vivo with the intracellular fluorescent dye carboxyfluorescein diacetate succinimidyl ester. Nature Protocols 2, 2049-2056 (2007).
    4. Parish, C. R. Fluorescent dyes for lymphocyte migration and proliferation studies. Immunol Cell Biol 77, 499-508 (1999).

    Comments

    13 Comments

    I've noticed that sometimes I don't get distinct peaks and I haven't figured out why that is the case. I've been thinking that perhaps viewing the cells under the microscope often after the cells have been stained could possibly alter the CFSE fluorescence. Do you have any tips to ensure distinct resolution of the proliferating cells? Thanks.
    Reply

    Posted by: AnonymousNovember 18, 2011, 11:01 AM

    One of the most important steps to get good resolution of fluorescence division peaks is to ensure you label cells as quickly as possible. Keep in mind that some cell populations give better resolution of fluorescence division peaks than others (eg B cells can give poorer resolution than T cells and homogeneous cell population have better resolution than heterogeneous populations). Viewing the cells briefly under a (light) microscope generally dŒs not drastically alter the resolution of fluorescence division peaks. If you need further help, please feel free to email me directly. Ben Quah
    Reply

    Posted by: AnonymousNovember 20, 2011, 6:10 PM

    CFSE stained cells were acquired at ohr and then ²4 hr and 36 hr.
    The mean at 0hr was arnd ²700 where as after ²4 hrs was aorund 1000.
    Y sucha drastic shift in the intensity of the CFSE population at the same voltages and the non labelled control.
    Plesae explain....
    Reply

    Posted by: AnonymousFebruary 4, 2012, 7:19 AM

    It is normal for CFSE-labeled cells to have a loss in fluorescence intensity over the initial ²4hrs. This loss stabilizes after the first day, although there still is a reduction in fluorescence intensity over time. Several factors might be responsible for this eg, loss of dye molecules from cells that are not attached to long lived proteins, turnover of labeled proteins ...etc
    Reply

    Posted by: AnonymousFebruary 5, 2012, 9:41 PM

    can CFSE be also used for analyzing the proliferation of any other cells like endothelial cells.
    Reply

    Posted by: AnonymousSeptember 18, 2012, 3:49 PM

    Yes, other cells have been shown to be amenable to CFSE-based proliferation analysis (e.g. hemopietic stem cells (Nordon, R.E., et al. British journal of haematology 1997; 98(3): 5²8-39.), myeloid leukemic progenitors (Holyoake, T., et al. Blood 1999; 94(6): ²056-64.) and bacteria (Ueckert, J.E., et al. Letters in applied microbiology 1997; ²5(4): ²95-9.)). CFSE labelling is essentially dependent on membrane permeability, intracellular esterase activity and amine reactivity so any cell meeting these requirements should label.

    However, the more heterogenous the population of cells is (endothelial cells for eg are quite heterogenous relative to lymphocytes), the more variable the initial fluorescence intensity is (ie the %CV of undivided cells for CFSE will be large (SD of >30% of MFI for eg)). This will make it very difficult to get clearly resolved division peaks with each successive division. So in this case you would get a general decrease in CFSE intensity as cells divide not discrete peaks, which still maybe useful to you (?).

    Good luck!

    Ben
    Reply

    Posted by: AnonymousSeptember 18, 2012, 7:55 PM

    Hi, I am a bioinformatician and I work on an algorithm to fit peaks over a CFSE/PKH²6 proliferation tracking experiments. Something similar to the ModFit software but openSource and Free. To complete the work I need some example files with CFSE and PKH²6 on lymphocytes. It is possible to get and use the experimental results from the Jove presentation (I need the FCS files).

    Best Regards.

    Davide Rambaldi (ieo.eu)
    Reply

    Posted by: Davide R.January 10, 2013, 11:15 AM

    i have a same problem whch already discussed here,i cant get the distinct peaks of cfse dye.how i got these peaks
    Reply

    Posted by: rabiya m.January 27, 2013, 4:15 AM

    Hi Rabiya,

    Sorry for the late reply, I have had issues logging on to this site.

    As mentioned above, one of the most important aspects to CFSE labeling for distinct peaks is to get rapid uniform labeling labeling of cells when CFDA, SE is first added to your cell suspension. The video here shows one way in which to achieve this (there are of course many others). If you are still getting problems with this, perhaps there are some other areas where something could be improved. Please read our Nature Protocols or Current Protocols in Immunology articles for more information on potential trouble shooting.

    I hope this helps,

    Ben
    Reply

    Posted by: AnonymousJune 7, 2013, 1:39 AM

    Hello,
    I an not geting proper peek with uniform peak for my samples. either I get two population or sometimes very low intensity. I dont understant what is the reason for this non-uniformity. I read somewhere that freezing-thawing the CFDE,SE stock destabilize it and so I feel that since I am using same stock every time, there might be some problem with CFDE,SE stock.. can please suggest me if I am right.
    Reply

    Posted by: farha m.March 8, 2013, 11:41 AM

    Hi Farha,

    Sorry for the late reply, I have had issues logging on to this site.

    I have been using freeze thawed stocks for some time now and I have not had any major issues with changes in uniformity of labeling. However, I am very careful not to let condensation form inside the stock vial since CFDA, SE will react in aqueous solution (i.e., make sure the stock vial equilibrates to room temperature before opening the vial and make sure your stock vials are sealed tightly, ideally with a screw top lid with a rubber seal).

    I hope this help,

    Ben
    Reply

    Posted by: AnonymousJune 7, 2013, 1:33 AM

    I want to know for how much time dŒs the staining could be detected and if this staining could diffused to other tissues.

    Reply

    Posted by: Lucia B.June 2, 2013, 1:26 PM

    Hi Lucia,

    CFSE labeled lymphocyte do decrease fluorescence over time, particularly after the first few days post labeling. After this period, however, the labeling is quite stable and loss may reflect the natural turn-over of proteins (and associated loss of the CFSE label). As a result, CFSE fluorescence can be detected weeks and even months after initial labeling, depending on how high your initial labeling concentration is.

    CFSE, typically, will not diffuse to significant levels to other tissues or cells (there is more on this in our recent paper published in the Journal of Immunological Methods Volume 379, Issues 1- ², Pages 1-14.)

    I hope this help,

    All the best,

    Ben
    Reply

    Posted by: AnonymousJune 7, 2013, 1:20 AM

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