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.

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:
<ol class="lalpha">
	<li>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.</li>
	<li>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).</li>
	<li>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.</li>
	<li>Resuspend 1.1 μL of the 5 mM stock of CFDA, SE in the 110 μL PBS.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
</ol>
2.2) Alternative method, which is also appropriate for higher cell numbers (10 - 30 x 107):
<ol class="lalpha">
	<li>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.</li>
	<li>Incubate the cells for 5 min at room temperature and wash as in steps 2.1(f) and 2.1(g).</li>
</ol>
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.
<img src="/files/ftp_upload/2259/2259fig1.jpg" alt="CFSE division analysis graph showing T cell proliferation in OVA-pulsed dendritic cells at varying doses." data-altupdatedat="1747911167000" data-alt="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.
<img src="/files/ftp_upload/2259/2259fig2.jpg" alt="Flow cytometry result; CD8 T-cell proliferation analysis; CFSE labeling; histogram plot." data-altupdatedat="1747911167000" data-alt="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.
<img src="/files/ftp_upload/2259/2259fig3.jpg" alt="Dye entry and protein labeling process diagram with CFDA-SE crossing plasma membrane, enzyme reaction." data-altupdatedat="1747911167000" data-alt="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.

<ol>
	<li>Lyons, A. B., Parish, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+lymphocyte+division+by+flow+cytometry.">Determination of lymphocyte division by flow cytometry.</a> J. Immunol. Methods. 171, 131-137 (1994).</li><li>Parish, C. R., Glidden, M. H., Quah, B. J., Warren, H. S., Coligan, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+the+intracellular+fluorescent+dye+CFSE+to+monitor+lymphocyte+migration+and+proliferation.">Use of the intracellular fluorescent dye CFSE to monitor lymphocyte migration and proliferation.</a> Current protocols in immunology. , (2009).</li><li>Quah, B. J., Warren, H. S., Parish, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+lymphocyte+proliferation+in+vitro+and+in+vivo+with+the+intracellular+fluorescent+dye+carboxyfluorescein+diacetate+succinimidyl+ester.">Monitoring lymphocyte proliferation in vitro and in vivo with the intracellular fluorescent dye carboxyfluorescein diacetate succinimidyl ester.</a> Nature Protocols. 2, 2049-2056 (2007).</li><li>Parish, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+dyes+for+lymphocyte+migration+and+proliferation+studies.">Fluorescent dyes for lymphocyte migration and proliferation studies.</a> Immunol Cell Biol. 77, 499-508 (1999).</li></ol>

No conflicts of interest declared.

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).

<table><tbody><tr><td>CFDA,SE</td><td>Molecular Probes, Life Technologies</td><td></td><td></td></tr><tr><td>DMSO</td><td>Cambridge Isotope Laboratories</td><td></td><td></td></tr><tr><td>Lymphocytes</td><td>eg mouse or human</td><td></td><td></td></tr><tr><td>RPMI</td><td>GIBCO, by Life Technologies</td><td></td><td></td></tr><tr><td>FCS</td><td>SAFC Global</td><td></td><td></td></tr><tr><td>10-15ml conical tubes</td><td>Falcon BD</td><td></td><td></td></tr><tr><td>PBS</td><td>GIBCO, by Life Technologies</td><td></td><td></td></tr><tr><td>Aluminium foil</td><td>Capral Aluminium</td><td></td><td></td></tr><tr><td>Vortex</td><td>Scientific Industries Inc.</td><td></td><td></td></tr><tr><td>Centrifuge</td><td>Eppendorf</td><td></td><td></td></tr><tr><td>Flow cytometer</td><td>BD Biosciences</td><td></td><td></td></tr></tbody></table>

the use of carboxyfluorescein diacetate succinimidyl ester (cfse) to monitor lymphocyte proliferation

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.

Watch this Scientific Journal Video about The Use of Carboxyfluorescein Diacetate Succinimidyl Ester CFSE to Monitor Lymphocyte Proliferation at JoVE.com

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

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.

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

Carboxyfluorescein succinimidyl ester (CFSE) is an effective and popular means to monitor lymphocyte division1-3.  CFSE covalently labels long-lived ...

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91.9K Views.  John Curtin School of Medical Research, Australian National University. CFSE labels covalent langlevende intracellulaire moleculen met de fluorescente kleurstof, carboxyfluoresceïne. Als zodanig, als een CFSE-gelabelde cel zich deelt, zijn nakomelingen hebben de helft van de fluorescentie, die daarmee kan worden gebruikt om de celdeling te beoordelen. Dit artikel beschrijft de procedures doorgaans gebruikt voor het labelen muis lymfocyten met CFSE.

Video: The Use of Carboxyfluorescein Diacetate Succinimidyl Ester CFSE to Monitor Lymphocyte Proliferation

Antigen Specific In Vivo Killing Assay using CFSE Labeled Target Cells

Carboxyfluorescein diacetate succinimidyl ester (CFSE) can be used to easily and quickly label a cell population of interest for in vivo investigation. This labeling has classically been used to study proliferation and migration. In the method presented here, we have shortened the timeline after adoptive transfer to look at survival and killing of epitope specific CFSE labeled target cells4-6. The level of specific killing of a CD8 + T cell clone can indicate the quality of the response, as their quantity may be misleading. Specific CD8+ T cells can become functionally exhausted over time with a decline in cytokine production and killing7,8. Also, certain CD8 + T cell clones may not kill as well as others with differing TCR specificities9. For effective Cell Mediated Immunity (CMI), antigens must be identified that produce not only adequate numbers of responding T cells, but also functionally robust responding T cells. Here we assess the percent cell specific killing of two peptide specific T cell clones in BALB/c mice.

Antigen Specific In Vivo Killing Assay using CFSE Labeled Target Cells

Many infections elicit a strong CTL response, but occasionally, the quantity of responding cells does not correlate to control of the pathogen1. One measure of CTL quality is their ability to kill specifically2. CFSE labeling of target cells can be used to investigate this CTL response quality in vivo3,4.

Carboxyfluorescein diacetate succinimidyl ester (CFSE) can be used to easily and quickly label a cell population of interest for in vivo investigation.  ...

Identification and Isolation of Slow-Dividing Cells in Human Glioblastoma Using Carboxy Fluorescein Succinimidyl Ester (CFSE)

Tumor heterogeneity represents a fundamental feature supporting tumor robustness and presents a central obstacle to the development of therapeutic strategies1. To overcome the issue of tumor heterogeneity, it is essential to develop assays and tools enabling phenotypic, (epi)genetic and functional identification and characterization of tumor subpopulations that drive specific disease pathologies and represent clinically relevant targets. It is now well established that tumors exhibit distinct sub-fractions of cells with different frequencies of cell division, and that the functional criteria of being slow cycling is positively associated with tumor formation ability in several cancers including those of the brain, breast, skin and pancreas as well as leukemia2-8. The fluorescent dye carboxyfluorescein succinimidyl ester (CFSE) has been used for tracking the division frequency of cells in vitro and in vivo in blood-borne tumors and solid tumors such as glioblastoma2,7,8. The cell-permeant non-fluorescent pro-drug of CFSE is converted by intracellular esterases into a fluorescent compound, which is retained within cells by covalently binding to proteins through reaction of its succinimidyl moiety with intracellular amine groups to form stable amide bonds9. The fluorescent dye is equally distributed between daughter cells upon divisions, leading to the halving of the fluorescence intensity with every cell division. This enables tracking of cell cycle frequency up to eight to ten rounds of division10. CFSE retention capacity was used with brain tumor cells to identify and isolate a slow cycling subpopulation (top 5% dye-retaining cells) demonstrated to be enriched in cancer stem cell activity2. 
This protocol describes the technique of staining cells with CFSE and the isolation of individual populations within a culture of human glioblastoma (GBM)-derived cells possessing differing division rates using flow cytometry2. The technique has served to identify and isolate a brain tumor slow-cycling population of cells by virtue of their ability to retain the CFSE labeling.

Identification and Isolation of Slow-Dividing Cells in Human Glioblastoma Using Carboxy Fluorescein Succinimidyl Ester CFSE

This video protocol demonstrates the application of the fluorescent dye carboxyfluorescein succinimidyl ester (CFSE) for the identification and separation of different sub-populations of cells in human glioblastoma based on frequency of cell division.

Tumor heterogeneity represents a fundamental feature supporting tumor robustness and presents a central obstacle to the development of therapeutic ...

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Functional Characterization of Regulatory Macrophages That Inhibit Graft-reactive Immunity

Macrophage accumulation in transplanted organs has long been recognized as a feature of allograft rejection1. Immunogenic monocytes infiltrate the allograft early after transplantation, mount a graft reactive response against the transplanted organ, and initiate organ rejection2. Recent data suggest that suppressive macrophages facilitate successful long-term transplantation3 and are required for the induction of transplantation tolerance4. This suggests a multidimensional concept of macrophage ontogeny, activation, and function, which demands a new roadmap for the isolation and analysis of macrophage function5. Due to the plasticity of macrophages, it is necessary to provide a methodology to isolate and characterize macrophages, depending on the tissue environment, and to define their functions according to different scenarios. Here, we describe a protocol for immune characterization of graft-infiltrating macrophages and the methods we used to functionally evaluate their capacity to inhibit CD8+ T proliferation and to promote CD4+Foxp3+ Treg expansion in vitro.

Macrophages are plastic cells of the hematopoietic system that have a crucial role in protective immunity and homeostasis. In this report, we describe optimized in vitro techniques to phenotypically and functionally characterize graft-infiltrating regulatory macrophages that accumulate in the transplanted organ under tolerogenic conditions.

Macrophage accumulation in transplanted organs has long been recognized as a feature of allograft rejection1. Immunogenic monocytes infiltrate the ...

Isolation and Activation of Murine Lymphocytes

B and T cells, with their extremely diverse antigen-receptor repertoires, have the ability to mount specific immune responses against almost any invading pathogen1,2. Understandably, such intricate abilities are controlled by a large number of molecules involved in various cellular processes to ensure timely and spatially regulated immune responses3. Here, we describe experimental procedures that allow rapid isolation of highly purified murine lymphocytes using magnetic cell sorting technology. The resulting purified lymphocytes can then be subjected to various in vitro or in vivo functional assays, such as the determination of lymphocyte signaling capacity upon stimulation by immunoblotting4 and the investigation of proliferative abilities by 3H-thymidine incorporation or carboxyfluorescein diacetate succinimidyl ester (CFSE) labeling5-7. In addition to comparing the functional capacities of control and genetically modified lymphocytes, we can also determine the T cell stimulatory capacity of antigen-presenting cells (APCs) in vivo, as shown in our representative results using transplanted CFSE-labeled OT-I T cells.

Lymphocytes are the major players in adaptive immune responses. Here, we present a lymphocyte purification protocol to determine the physiological functions of the desired molecules in lymphocyte activation in vitro and in vivo. The described experimental procedures are suitable for comparing functional capacities between control and genetically modified lymphocytes.

B and T cells, with their extremely diverse antigen-receptor repertoires, have the ability to mount specific immune responses against almost any invading ...

In Vivo Assay for Detection of Antigen-specific T-cell Cytolytic Function Using a Vaccination Model

Current methodologies for antigen-specific killing are limited to in vitro use or utilized in infectious disease models. However, there is not a protocol specifically intended to measure antigen-specific killing without an infection. This protocol is designed and describes methods to overcome these limitations by allowing for the detection of antigen-specific killing of a target cell by CD8+ T cells in vivo. This is accomplished by merging a vaccination model with a traditional CFSE-labeled target killing assay. This combination allows the researcher to assess the antigen-specific CTL potential directly and quickly as the assay is not dependent upon tumor growth or infection. In addition, the readout is based on flow cytometry and so should be readily accessible to most researchers. The major limitation of the study is identifying the timeline in vivo that is appropriate to the hypothesis being tested. Variations in antigen strength and mutations in the T cells that may result in differential cytolytic function need to be carefully assessed to determine the optimal time for cell harvest and assessment. The appropriate concentration of peptide for vaccination has been optimized for hgp10025-33 and OVA257-264, but further validation would be needed for other peptides that may be more appropriate to a given study. Overall, this protocol allows a quick assessment of killing function in vivo and can be adapted to any given antigen.

In Vivo Assay for Detection of Antigen-specific T-cell Cytolytic Function Using a Vaccination Model

The goal of this protocol is to allow for detection of in vivo antigen-specific killing of a target cell in a murine model.

Current methodologies for antigen-specific killing are limited to in vitro use or utilized in infectious disease models. However, there is not a protocol ...

Fluorescent Dye Labeling of Erythrocytes and Leukocytes for Studying the Flow Dynamics in Mouse Retinal Circulation

The retinal and choroidal blood flow dynamics may provide insight into the pathophysiology and sequelae of various ocular diseases, such as glaucoma, diabetic retinopathy, age-related macular degeneration (AMD) and other ocular inflammatory conditions. It may also help to monitor the therapeutic responses in the eye. The proper labeling of the blood cells, coupled with live-cell imaging of the labeled cells, allows for the investigation of the flow dynamics in the retinal and choroidal circulation. Here, we describe the standardized protocols of 1.5% indocyanine green (ICG) and 1% sodium fluorescein labeling of mice erythrocytes and leukocytes, respectively. Scanning laser ophthalmoscopy (SLO) was applied to visualize the labeled cells in the retinal circulation of C57BL/6J mice (wild type). Both methods demonstrated distinct fluorescently labeled cells in the mouse retinal circulation. These labeling methods can have wider applications in various ocular disease models.

Live-cell imaging of the labeled blood cells in ocular circulation can provide information about inflammation and ischemia in diabetic retinopathy and age-related macular degeneration. A protocol to label blood cells and image the labeled cells in the retinal circulation is described.

The retinal and choroidal blood flow dynamics may provide insight into the pathophysiology and sequelae of various ocular diseases, such as glaucoma, ...

Quantification of Proliferating Human Antigen-specific CD4+ T Cells using Carboxyfluorescein Succinimidyl Ester

Described is a simple, in vitro, dye dilution-based method for measuring antigen-specific CD4+ T cell proliferation in human peripheral blood mononuclear cells (PBMCs). The development of stable, non-toxic, fluorescent dyes such as carboxyfluorescein succinimidyl ester (CFSE) allows for rare, antigen-specific T cells to be distinguished from bystanders by diminution in fluorescent staining, as detected by flow cytometry. This method has the following advantages over alternative approaches: (i) it is very sensitive to low-frequency T cells, (ii) no knowledge of the antigen or epitope is required, (iii) the phenotype of the responding cells can be analyzed, and (iv) viable, responding cells can be sorted and used for further analysis, such as T cell cloning.

Quantification of Proliferating Human Antigen-specific CD4+ T Cells using Carboxyfluorescein Succinimidyl Ester

Presented here is a protocol for measuring proliferating CD4+ T cells in response to antigenic proteins or peptides using dye dilution. This assay is particularly sensitive to rare antigen-specific T cells and can be modified to facilitate cloning of antigen-specific cells.

Described is a simple, in vitro, dye dilution-based method for measuring antigen-specific CD4+ T cell proliferation in human peripheral blood mononuclear ...

Rapid In Vivo Assessment of Adjuvant's Cytotoxic T Lymphocytes Generation Capabilities for Vaccine Development

The assessment of modern sub-unit vaccines reveals that the generation of neutralizing antibodies is important but not sufficient for adjuvant selection. Therefore, adjuvants with both humoral and cellular immuno-stimulatory capabilities that are able to promote cytotoxic T lymphocytes (CTL) responses are urgently needed. Thus, faithful monitoring of adjuvant candidates that induce cross-priming and subsequently enhance CTL generation represents a crucial step in vaccine development. In here we present an application for a method that uses SIINFEKL-specific (OT-I) T cells to monitor the cross-presentation of the model antigen ovalbumin (OVA) in vivo in the presence of different adjuvant candidates. This method represents a rapid test to select adjuvants with the best cross-priming capabilities. The proliferation of CD8+ T cells is the most valuable indication of cross-priming and it is also regarded as a correlate of adjuvant-induced cross-presentation. This feature can be evaluated in different immune organs like lymph nodes and spleen. The extent of the CTL generation can also be monitored, thereby giving insights on the nature of a local (draining lymph node mainly) or a systemic response (distant lymph nodes and/or spleen). This technique further allows multiple modifications for testing drugs that can inhibit specific cross-presentation pathways and also offers the possibility to be used in different strains of conventional and genetically modified mice. In summary, the application that we present here will be useful for vaccine laboratories in industry or academia that develop or modify chemical adjuvants for vaccine research and development.

Rapid In Vivo Assessment of Adjuvant's Cytotoxic T Lymphocytes Generation Capabilities for Vaccine Development

We present here an application for a standard immunological technique (CFSE stained OT-I proliferation) intended to rapidly monitor adjuvant-mediated cytotoxic T lymphocyte (CTL) generation in vivo. This fast estimation of CTL capacities is useful for the development of prophylactic vaccines against intracellular pathogens as well as therapeutic cancer vaccines.

The assessment of modern sub-unit vaccines reveals that the generation of neutralizing antibodies is important but not sufficient for adjuvant selection. ...

Cell Cycle Analysis: Assessing CD4 and CD8 T Cell Proliferation After Stimulation Using CFSE Staining and Flow Cytometry

Source: Perchet Thibaut1,2,3, Meunier Sylvain1,2,3, Sophie Novault4, Rachel Golub1,2,3 
1 Unit for Lymphpoiesis, Department of Immunology, Pasteur Institute, Paris, France 
2 INSERM U1223, Paris, France 
3 Universit&#233; Paris Diderot, Sorbonne Paris Cit&#233;, Cellule Pasteur, Paris, France 
4 Flow Cytometry Platfrom, Cytometry and Biomarkers UtechS, Center for Translational Science, Pasteur Institute, Paris, France
The cell cycle is a universal process of life. During the cell cycle, a cell undergoes several modifications to divide into two daughter cells. This mechanism occurs throughout an organism&#39;s life in response to its needs. Cell divisions and embryonic development produce a full organism from a single-celled zygote. During adulthood, the cell cycle is central to many critical biological processes, such as tissue repairs.
Mechanisms of cell division are tightly controlled events where the cell undergoes stepwise modifications before final division. Cells that are not yet in the cycle are described as being in the Gap 0 (G0) phase. During this stage the cell is considered quiescent. When the cell starts to cycle, four distinct phases are recognized: Gap 1 (G1), Synthesis (S), Gap 2 (G2) and Mitosis (M). G1 phase is a checkpoint for resources needed by the cell for DNA synthesis. Then, S phase occurs, and DNA replication starts, followed by the G2 interphase, another checkpoint that controls all elements necessary to for the cell to divide. Finally, the cell enters mitosis and divides into two daughter cells.
Cell division is a highly informative parameter in many different biological systems. In the field of immunology, analysis of leukocyte proliferation can indicate the mechanism of the immune response. Other domains of investigation also rely on cell cycle analysis. For instance, analysis of the cell cycle during tumor development has improved our understanding of cancer.
Many fluorescent dyes are now available for tracking cell proliferation. These dyes differ in their chemical and spectral properties. Two different classes of dyes exist: protein dyes permanently combine with protein by forming a covalent bond, and membrane dyes stably intercalate within cell membranes via strong hydrophobic associations. In vitro and in vivo studies of immune cell proliferation by flow cytometry are among the most common applications of both classes of cell tracking dyes (1, 2).
CFSE (Carboxyfluorescein succinimidyl ester) is a fluorescent dye that marks dividing cells. Initially, all the cells receive the same amount of dye; dividing cells evenly split the dye they received between their two daughter cells. Consequently, the cell cycle can be followed by the progressive decrease of dye intensity in the cells. CFSE staining is followed by conventional multi-parametric flow cytometry, a high-throughput, fluorescence-based technology that allows phenotypic and functional characterization of cells based on their degree of CFSE staining (3).
In the following experiment, we assess the proliferation of CD4+ and CD8+ T cells in-vitro, following CD3 stimulation, using CFSE staining and flow cytometry.

Cell Cycle Analysis: Assessing CD4 and CD8 T Cell Proliferation

Source: Perchet Thibaut1,2,3, Meunier Sylvain1,2,3, Sophie Novault4, Rachel Golub1,2,3
1 Unit for Lymphpoiesis, Department of Immunology, Pasteur ...

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An In Vitro Assay for Evaluating the Immunomodulatory Impact of Monocytes on Leukocyte Proliferation

Source: Hsu, P., et al. <a href="https://app.jove.com/v/53265">Assessment of the Immunomodulatory Properties of Human Mesenchymal Stem Cells (MSCs).</a> J. Vis. Exp. (2015)
This video demonstrates an in vitro assay to explore the immunomodulatory influence of mesenchymal stem cells (MSCs) on effector T cell proliferation. The assay includes co-culturing MSCs with PBMCs to induce the monocyte population to acquire an immunomodulatory phenotype, leading to the secretion of interleukin-10 (IL-10). Following incubation with activated CD4+ T cells, the induced monocytes effectively suppress T cell proliferation.

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Density Gradient Isolation of ...

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

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