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 &mu;L aliquots at -20 &deg;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 &mu;L 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 &mu;L of the 5 mM stock of CFDA, SE in the 110 &mu;L 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 &mu;M, 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 &deg;C PBS containing 5 % HI FCS, sedimenting by centrifugation at 300 x g for 5 min at 20 &deg;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 &mu;M) CFDA, SE solution by adding 2 &mu;L 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 &deg;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 &mu;g 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 &mu;g 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="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="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="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).

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		<th>Type</th>
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		<th>Catalogue Number</th>
		<th>Comment</th>
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<td>CFDA,SE</td>
<td>&nbsp;</td>
<td>Molecular Probes</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>DMSO</td>
<td>&nbsp;</td>
<td>Cambridge Isotope Laboratories Inc.</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Lymphocytes</td>
<td>&nbsp;</td>
<td>eg mouse or human</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>RPMI</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>FCS</td>
<td>&nbsp;</td>
<td>SAFC Biosciences</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>10-15ml conical tubes</td>
<td>&nbsp;</td>
<td>Falcon, Becton Dickinson</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>PBS</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>Aluminium foil</td>
<td>&nbsp;</td>
<td>Capral Aluminium</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>Vortex</td>
<td>&nbsp;</td>
<td>Scientific Industries</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Centrifuge</td>
<td>&nbsp;</td>
<td>Eppendorf (5810 R)</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Flow cytometer</td>
<td>&nbsp;</td>
<td>LSR-II (BD Biosciences)</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
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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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91.3K Views.  John Curtin School of Medical Research, Australian National University. 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.

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

Finger-stick Blood Sampling Methodology for the Determination of Exercise-induced Lymphocyte Apoptosis

Exercise is a physiological stimulus capable of inducing apoptosis in immune cells. To date, various limitations have been identified with the measurement of this phenomenon, particularly relating to the amount of time required to isolate and treat a blood sample prior to the assessment of cell death. Because of this, it is difficult to determine whether reported increases in immune cell apoptosis can be contributed to the actual effect of exercise on the system, or are a reflection of the time and processing necessary to eventually obtain this measurement. In this article we demonstrate a rapid and minimally invasive procedure for the analysis of exercise-induced lymphocyte apoptosis. Unlike other techniques, whole blood is added to an antibody panel immediately upon obtaining a sample. Following the incubation period, red blood cells are lysed and samples are ready to be analyzed. The use of a finger-stick sampling procedure reduces the volume of blood required, and minimizes the discomfort to subjects.

Exercise is capable of inducing apoptosis in immune cells. There are various measurement limitations, particularly relating to the amount of time required to isolate and treat a blood sample prior to the assessment. Demonstrated is a rapid and minimally invasive procedure for the analysis of exercise-induced lymphocyte apoptosis.

Exercise is a physiological stimulus capable of inducing apoptosis in immune cells. To date, various limitations have been identified with the measurement ...

A Visual Assay to Monitor T6SS-mediated Bacterial Competition

Type VI secretion systems (T6SSs) are molecular nanomachines allowing Gram-negative bacteria to transport and inject proteins into a wide variety of target cells1,2. The T6SS is composed of 13 core components and displays structural similarities with the tail-tube of bacteriophages3. The phage uses a tube and a puncturing device to penetrate the cell envelope of target bacteria and inject DNA. It is proposed that the T6SS is an inverted bacteriophage device creating a specific path in the bacterial cell envelope to drive effectors and toxins to the surface. The process could be taken further and the T6SS device could perforate other cells with which the bacterium is in contact, thus injecting the effectors into these targets. The tail tube and puncturing device parts of the T6SS are made with Hcp and VgrG proteins, respectively4,5.
The versatility of the T6SS has been demonstrated through studies using various bacterial pathogens. The Vibrio cholerae T6SS can remodel the cytoskeleton of eukaryotic host cells by injecting an "evolved" VgrG carrying a C-terminal actin cross-linking domain6,7. Another striking example was recently documented using Pseudomonas aeruginosa which is able to target and kill bacteria in a T6SS-dependent manner, therefore promoting the establishment of bacteria in specific microbial niches and competitive environment8,9,10.
In the latter case, three T6SS-secreted proteins, namely Tse1, Tse2 and Tse3 have been identified as the toxins injected in the target bacteria (Figure 1). The donor cell is protected from the deleterious effect of these effectors via an anti-toxin mechanism, mediated by the Tsi1, Tsi2 and Tsi3 immunity proteins8,9,10. This antimicrobial activity can be monitored when T6SS-proficient bacteria are co-cultivated on solid surfaces in competition with other bacterial species or with T6SS-inactive bacteria of the same species8,11,12,13.
The data available emphasized a numerical approach to the bacterial competition assay, including time-consuming CFU counting that depends greatly on antibiotic makers. In the case of antibiotic resistant strains like P. aeruginosa, these methods can be inappropriate. Moreover, with the identification of about 200 different T6SS loci in more than 100 bacterial genomes14, a convenient screening tool is highly desirable. We developed an assay that is easy to use and requires standard laboratory material and reagents. The method offers a rapid and qualitative technique to monitor the T6SS-dependent bactericidal/bacteriostasis activity by using a reporter strain as a prey (in this case Escherichia coli DH5&alpha;) allowing a-complementation of the lacZ gene. Overall, this method is graphic and allows rapid identification of T6SS-related phenotypes on agar plates. This experimental protocol may be adapted to other strains or bacterial species taking into account specific conditions such as growth media, temperature or time of contact.

We describe a qualitative assay to monitor bacterial competition mediated by the Pseudomonas aeruginosa type VI secretion system (T6SS). The assay relies on the survival/killing of Escherichia coli target cells carrying a lacZ-reporter. This technique is adjustable to assess the bactericidal/bacteriostasis activity of T6SS-proficient microorganisms.

Type VI secretion systems (T6SSs) are molecular  nanomachines allowing Gram-negative bacteria to transport and inject proteins  into a wide variety of ...

Use of In vivo Imaging to Monitor the Progression of Experimental Mouse Cytomegalovirus Infection in Neonates

Human Cytomegalovirus (HCMV or HHV-5) is a life-threatening pathogen in immune-compromised individuals. Upon congenital or neonatal infection, the virus can infect and replicate in the developing brain, which may induce severe neurological damage, including deafness and mental retardation. Despite the potential severity of the symptoms, the therapeutic options are limited by the unavailability of a vaccine and the absence of a specific antiviral therapy. Furthermore, a precise description of the molecular events occurring during infection of the central nervous system (CNS) is still lacking since observations mostly derive from the autopsy of infected children. Several animal models, such as rhesus macaque CMV, have been developed and provided important insights into CMV pathogenesis in the CNS. However, despite its evolutionary proximity with humans, this model was limited by the intracranial inoculation procedure used to infect the animals and consistently induce CNS infection. Furthermore, ethical considerations have promoted the development of alternative models, among which neonatal infection of newborn mice with mouse cytomegalovirus (MCMV) has recently led to significant advances. For instance, it was reported that intraperitoneal injection of MCMV to Balb/c neonates leads to infection of neurons and glial cells in specific areas of the brain. These findings suggested that experimental inoculation of mice might recapitulate the deficits induced by HCMV infection in children. Nevertheless, a dynamic analysis of MCMV infection of neonates is difficult to perform because classical methodology requires the sacrifice of a significant number of animals at different time points to analyze the viral burden and/or immune-related parameters. To circumvent this bottleneck and to enable future investigations of rare mutant animals, we applied in vivo imaging technology to perform a time-course analysis of the viral dissemination in the brain upon peripheral injection of a recombinant MCMV expressing luciferase to C57Bl/6 neonates.

Use of In vivo Imaging to Monitor the Progression of Experimental Mouse Cytomegalovirus Infection in Neonates

Human Cytomegalovirus (HCMV) infection of neonates represents an important cause of mental retardation, yet the molecular events leading to virus-induced pathogenesis are still poorly understood. To investigate the dynamics of brain infection, we adapted whole-animal in vivo imaging to perform time-course analysis of neonates infected with a luciferase-recombinant virus.

Human Cytomegalovirus (HCMV or HHV-5) is a life-threatening  pathogen in immune-compromised individuals. Upon congenital or neonatal  infection, the virus ...

Using Fluorescent Proteins to Monitor Glycosome Dynamics in the African Trypanosome

Trypanosoma brucei is a kinetoplastid parasite that causes human African trypanosomiasis (HAT), or sleeping sickness, and a wasting disease, nagana, in cattle1. The parasite alternates between the bloodstream of the mammalian host and the tsetse fly vector. The composition of many cellular organelles changes in response to these different extracellular conditions2-5.
Glycosomes are highly specialized peroxisomes in which many of the enzymes involved in glycolysis are compartmentalized. Glycosome composition changes in a developmental and environmentally regulated manner4-11. Currently, the most common techniques used to study glycosome dynamics are electron and fluorescence microscopy; techniques that are expensive, time and labor intensive, and not easily adapted to high throughput analyses.
To overcome these limitations, a fluorescent-glycosome reporter system in which enhanced yellow fluorescent protein (eYFP) is fused to a peroxisome targeting sequence (PTS2), which directs the fusion protein to glycosomes12, has been established. Upon import of the PTS2eYFP fusion protein, glycosomes become fluorescent. Organelle degradation and recycling results in the loss of fluorescence that can be measured by flow cytometry. Large numbers of cells (5,000 cells/sec) can be analyzed in real-time without extensive sample preparation such as fixation and mounting. This method offers a rapid way of detecting changes in organelle composition in response to fluctuating environmental conditions.

Glycosome dynamics in African trypanosomes are difficult to study by traditional cell biology techniques such as electron and fluorescence microscopy. As a means of observing dynamic organelle behavior, a fluorescent-organelle reporter system has been used in conjunction with flow cytometry to monitor real-time glycosome dynamics in live parasites.

Trypanosoma brucei is a kinetoplastid parasite that causes human African trypanosomiasis (HAT), or sleeping sickness, and a wasting disease, nagana, in ...

Immunofluorescence to Monitor the Cellular Uptake of Human Lactoferrin and its Associated Antiviral Activity Against the Hepatitis C Virus

Immunofluorescence is a laboratory technique commonly used to study many aspects of biology. It is typically used to visualize the distribution and/or localization of a target molecule in cells and tissues. Immunofluorescence relies on the specificity of fluorescent-labelled antibodies against their corresponding antigens within a cell. Both direct and indirect immunofluorescence approaches can be used which rely on the use of antibodies linked with a fluorochrome. Direct immunofluorescence is less frequently used because it provides lower signal, involves higher cost and less flexibility. In contrast, indirect immunofluorescence is more commonly used because of its high sensitivity and provides an amplified signal since more than one secondary antibody can attach to each primary antibody. In this manuscript, both epifluorescence microscopy and confocal microscopy were used to monitor the internalization of human lactoferrin, an important component of the immune system, into hepatic cells. Moreover, we monitored the inhibitory potential of hLF on the intracellular replication of the Hepatitis C virus using immunofluorescence. Both the advantages and disadvantages associated with these approaches are discussed.

The human lactoferrin (hLF) is a component of the immune system. In this study, immunofluorescence assays are used to demonstrate both the hepatocellular uptake of hLF and a qualitative reduction in the hepatitis C virus replication upon treatment with hLF.

Immunofluorescence is a laboratory technique commonly used to study many aspects of biology. It is typically used to visualize the distribution and/or ...

The Use of Flow Cytometry to Assess the State of Chromatin in T Cells

During a proper immune response, quiescent T cells become activated upon antigen presentation to their antigen-specific T cell receptor. This leads to clonal proliferation of only those T cells that bear a receptor that recognizes the antigen. Chromatin decondensation is a hallmark of T cell activation and is required for T cells to acquire the ability to proliferate after antigen engagement. This change in chromatin condensation can be detected using antibodies raised against histone proteins. These antibodies cannot bind to their epitopes in na&#239;ve T cells as well as they can in activated T cells. We describe how to simultaneously stain T cell-specific surface markers, track viability with a fixable dead cell stain, and measure chromatin status via intracellular staining of Histone H3 proteins. Stained cells are analyzed by flow cytometry and chromatin condensation status is measured as the mean fluorescence intensity (MFI) of the Histone H3 stain. Chromatin decondensation during T cell activation is demonstrated as an increase in the MFI

Flow cytometry can be utilized to assess the state of chromatin within T cells. This protocol allows scientists to interpret evidence of chromatin decondensation during T cell activation demonstrated by an increase in the mean florescence intensity (MFI) of fluorescent Histone H3 antibodies

During a proper immune response, quiescent T cells become activated upon antigen presentation to their antigen-specific T cell receptor. This leads to ...

Assay of Adhesion Under Shear Stress for the Study of T Lymphocyte-Adhesion Molecule Interactions

Overall, T cell adhesion is a critical component of function, contributing to the distinct processes of cellular recruitment to sites of inflammation and interaction with antigen presenting cells (APC) in the formation of immunological synapses. These two contexts of T cell adhesion differ in that T cell-APC interactions can be considered static, while T cell-blood vessel interactions are challenged by the shear stress generated by circulation itself. T cell-APC interactions are classified as static in that the two cellular partners are static relative to each other. Usually, this interaction occurs within the lymph nodes. As a T cell interacts with the blood vessel wall, the cells arrest and must resist the generated shear stress.1,2 These differences highlight the need to better understand static adhesion and adhesion under flow conditions as two distinct regulatory processes. The regulation of T cell adhesion can be most succinctly described as controlling the affinity state of integrin molecules expressed on the cell surface, and thereby regulating the interaction of integrins with the adhesion molecule ligands expressed on the surface of the interacting cell. Our current understanding of the regulation of integrin affinity states comes from often simplistic in vitro model systems. The assay of adhesion using flow conditions described here allows for the visualization and accurate quantification of T cell-epithelial cell interactions in real time following a stimulus. An adhesion under flow assay can be applied to studies of adhesion signaling within T cells following treatment with inhibitory or stimulatory substances. Additionally, this assay can be expanded beyond T cell signaling to any adhesive leukocyte population and any integrin-adhesion molecule pair.

This flow adhesion assay provides a simple, high impact model of T cell-epithelial cell interactions. A syringe pump is used to generate shear stress, and confocal microscopy captures images for quantification. The goal of these studies is to effectively quantify T cell adhesion using flow conditions.

Overall, T cell adhesion is a critical component of function, contributing to the distinct processes of cellular recruitment to sites of inflammation and ...

Detection of Trypanosoma brucei Variant Surface Glycoprotein Switching by Magnetic Activated Cell Sorting and Flow Cytometry

Trypanosoma brucei, a protozoan parasite that causes both Human and Animal African Trypanosomiasis (known as sleeping sickness and nagana, respectively) cycles between a tsetse vector and a mammalian host. It evades the mammalian host immune system by periodically switching the dense, variant surface glycoprotein (VSG) that covers its surface. The detection of antigenic variation in Trypanosoma brucei can be both cumbersome and labor intensive. Here, we present a method for quantifying the number of parasites that have 'switched' to express a new VSG in a given population. The parasites are first stained with an antibody against the starting VSG, and then stained with a secondary antibody attached to a magnetic bead. Parasites expressing the starting VSG are then separated from the rest of the population by running the parasites over a column attached to a magnet. Parasites expressing the dominant, starting VSG are retained on the column, while the flow-through contains parasites that express a new VSG as well as some contaminants expressing the starting VSG. This flow-through population is stained again with a fluorescently labeled antibody against the starting VSG to label contaminants, and propidium iodide (PI), which labels dead cells. A known number of absolute counting beads that are visible by flow cytometry are added to the flow-through population. The ratio of beads to number of cells collected can then be used to extrapolate the number of cells in the entire sample. Flow cytometry is used to quantify the population of switchers by counting the number of PI negative cells that do not stain positively for the starting, dominant VSG. The proportion of switchers in the population can then be calculated using the flow cytometry data.

Detection of Trypanosoma brucei Variant Surface Glycoprotein Switching by Magnetic Activated Cell Sorting and Flow Cytometry

African trypanosomes grown in vitro undergo antigenic variation at a low rate, such that populations are made up of parasites expressing a dominant variant surface glycoprotein (VSG) type and a small population of "switched" variants. This protocol describes a fast method for detecting and quantifying these populations.

Trypanosoma brucei, a protozoan parasite that causes both Human and Animal African Trypanosomiasis (known as sleeping sickness and nagana, respectively) ...

Development of a Hepatitis B Virus Reporter System to Monitor the Early Stages of the Replication Cycle

Currently, it is possible to construct recombinant forms of various viruses, such as human immunodeficiency virus 1 (HIV-1) and hepatitis C virus (HCV), that carry foreign genes such as a reporter or marker protein in their genomes. These recombinant viruses usually faithfully mimic the life cycle of the original virus in infected cells and exhibit the same host range dependence. The development of a recombinant virus enables the efficient screening of inhibitors and the identification of specific host factors. However, to date the construction of recombinant hepatitis B virus (HBV) has been difficult because of various experimental limitations. The main limitation is the compact genome size of HBV, and a fairly strict genome size that does not exceed 1.3 genome sizes, that must be packaged into virions. Thus, the size of a foreign gene to be inserted should be smaller than 0.4 kb if no deletion of the genome DNA is to be performed. Therefore, to overcome this size limitation, the deletion of some HBV DNA is required. Here, we report the construction of recombinant HBV encoding a reporter gene to monitor the early stage of the HBV replication cycle by replacing part of the HBV core-coding region with the reporter gene by deleting part of the HBV pol coding region. Detection of recombinant HBV infection, monitored by the reporter activity, was highly sensitive and less expensive than detection using the currently available conventional methods to evaluate HBV infection. This system will be useful for a number of applications including high-throughput screening for the identification of anti-HBV inhibitors, host factors and virus-susceptible cells.

Here we describe a newly developed hepatitis B virus (HBV) reporter system to monitor the early stages of the HBV life cycle. This simplified in vitro system will aid in the screening of anti-HBV agents using a high-throughput strategy.

Currently, it is possible to construct recombinant forms of various viruses, such as human immunodeficiency virus 1 (HIV-1) and hepatitis C virus (HCV), ...

Dextran Enhances the Lentiviral Transduction Efficiency of Murine and Human Primary NK Cells

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining the role of the gene of interest in the development, differentiation, and function of NK cells. Recent advances related to chimeric antigen receptors (CARs) in cancer immunotherapy accentuate the need for an efficient method to deliver exogenous genes to effector lymphocytes. The efficiencies of lentiviral-mediated gene transductions into primary human or mouse NK cells remain significantly low, which is a major limiting factor. Recent advances using cationic polymers, such as polybrene, show an improved gene transduction efficiency in T cells. However, these products failed to improve the transduction efficiencies of NK cells. This work shows that dextran, a branched glucan polysaccharide, significantly improves the transduction efficiency of human and mouse primary NK cells. This highly reproducible transduction methodology provides a competent tool for transducing human primary NK cells, which can vastly improve clinical gene delivery applications and thus NK cell-based cancer immunotherapy.

The goal of this study was to formulate technologies that allow for successful gene transduction in primary natural killer (NK) cells. The dextran-mediated lentiviral transduction of human or mouse primary NK cells results in higher gene expression efficiencies. This method of gene transduction will vastly improve NK cell genetic manipulation.

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining ...