Dye Carboxyfluorescein Diacetate Succinimidyl Ester Research Articles

HIV Infection Elicits Differential Transcriptomic Remodeling in CD4+ T Cells with Variable Proliferative Responses to the T Cell Receptor Stimulus.

Identification of a cellular biomarker of latent HIV infection will facilitate the latent reservoir detection, quantification, and targeting for elimination. Unfortunately, the latency biomarkers reported in the literature define only a fraction of the entire reservoir. The latent HIV reservoir may be established in dividing cells that subsequently return to quiescence and in resting cells. The strength of the T cell receptor (TCR) signaling at the time of infection affects characteristics of the established reservoir, such as the ability to reactivate with latency reversing agents. To better understand the cellular environments before latency establishment, we characterized transcriptomic remodeling induced by the initial HIV infection in cells with differential proliferative responses to the TCR stimulus. Cell proliferation was monitored using the viable dye carboxyfluorescein diacetate succinimidyl ester. Cells that divided many times, a few times, or remained non-dividing were subjected to single-cell RNA sequencing. A subset of identified transcriptional changes induced by HIV infection was independent of the number of cell divisions; however, responses unique to different cell subsets were also detected. Some of these early gene expression changes were consistent with reported markers of latently infected cells. We pose that the latency biomarkers may depend on the cellular proliferative state at the time of infection.

The ATF6-EGF Pathway Mediates the Awakening of Slow-Cycling Chemoresistant Cells and Tumor Recurrence by Stimulating Tumor Angiogenesis.

Slow-cycling cancer cells (SCCs) with a quiescence-like phenotype are believed to perpetrate cancer relapse and progression. However, the mechanisms that mediate SCC-derived tumor recurrence are poorly understood. Here, we investigated the mechanisms underlying cancer recurrence after chemotherapy, focusing on the interplay between SCCs and the tumor microenvironment. We established a preclinical model of SCCs by exposing non-small-cell lung cancer (NSCLC) cells to either the proliferation-dependent dye carboxyfluorescein diacetate succinimidyl ester (CFSE) or chemotherapeutic drugs. An RNA sequencing analysis revealed that the established SCCs exhibited the upregulation of a group of genes, especially epidermal growth factor (EGF). Increases in the number of vascular endothelial growth factor receptor (VEGFR)-positive vascular endothelial cells and epidermal growth factor receptor (EGFR) activation were found in NSCLC cell line- and patient-derived xenograft tumors that progressed upon chemotherapy. EGFR tyrosine kinase inhibitors effectively suppressed the migration and tube formation of vascular endothelial cells. Furthermore, activating transcription factor 6 (ATF6) induced the upregulation of EGF, and its antagonism effectively suppressed these SCC-mediated events and inhibited tumor recurrence after chemotherapy. These results suggest that the ATF6-EGF signaling axis in SCCs functions to trigger the angiogenesis switch in residual tumors after chemotherapy and is thus a driving force for the switch from SCCs to actively cycling cancer cells, leading to tumor recurrence.

Detection and Quantification of Extracellular Vesicles via FACS: Membrane Labeling Matters!

The field of extracellular vesicle (EV) research is challenged by the lack of standardized protocols to identify and specifically distinguish between exosomes and ectosomes, which are released via exocytosis or plasma membrane shedding, respectively. Using sequential centrifugation, we separated EV subpopulations from supernatants of COLO 357 pancreas carcinoma cells based on size and mass. After 10,000× g centrifugation, we reconstituted high-speed (hs) EVs from the pellet, directly labeled them with the membrane dye carboxyfluorescein diacetate succinimidyl ester (CFSE), and performed flow cytometry based analysis. The aim was to optimize the conditions for EV labeling and detection and hence to obtain a maximum yield of intact hsEVs. We found that, for sufficient labeling of EVs, minimal temperature variations and short incubation times correlated with EV stability. Furthermore, threshold adjustment significantly improved the sensitivity of the flow cytometer for the detection of CFSE labeled hsEVs. When cells were CFSE labeled, we observed a transition of fluorescence onto EVs that were reconstituted from the pellet but not onto those that remained in the supernatant after hs centrifugation, suggesting the indirect labeling of EVs based on the way of biogenesis as a specific method for the distinction of exosomes and ectosomes. Protocol standardization is of major importance for the use of EVs as diagnostic markers in liquid biopsies.

Rosemary has immunosuppressant activity mediated through the STAT3 pathway

ObjectiveIn Europe extracts of Rosmarinus officinalis were traditionally used for the treatment of rheumatic diseases. We investigated the capacity of standardized aqueous extracts of Rosmarinus officinalis on human primary lymphocyte function in vitro, as activated lymphocytes are an important mediator of rheumatic diseases. MethodsLymphocyte proliferation was measured using membrane-permeable dye carboxyfluorescein diacetate succinimidyl ester (CFSE). Apoptosis was analysed by surface staining of phosphatidylserine (annexin V-assay) and necrosis was analysed by staining with propidium iodide. Modification of cell activity was detected by surface staining of CD69 and CD25. The activity of STAT3 in T-lymphocytes was determined by intracellular staining of STAT3 molecules. All endpoints were analyzed by using flow cytometry. The Rosmarinus officinalis extract was investigated at concentrations of 0.05–25 mg/mL. Analysis of the extract was performed using HPLC methods. ResultsRosmarinus officinalis inhibited proliferation of human lymphocytes and CD4+ T-cells in a dose-dependent manner (3.1–25 mg/mL) through induction of apoptosis. The intracellular signalling pathway STAT3 in T-cells, but not NF-kappaB and ERK1/2 in T- and B-cells was inhibited in a dose-dependent manner by Rosmarinus officinalis (0.2-6.2 mg/mL). Rosmanol, carnosolic acid, carnosol and trans-caffeic acid were tested in the same cellular models as the crude extract. From these, only trans-caffeic acid inhibited lymphocyte proliferation and STAT3 (30–100 μg/mL). Trans-caffeic acid was found in the extract in a concentration of 14.7 μg/mL. ConclusionsWe conclude that an immunosuppressive effect of Rosmarinus officinalis is mostly due to the effect of trans-caffeic acid. It results in inhibition of the activity of STAT3 causing induction of apoptosis and inhibition of proliferation of T-lymphocytes.

Transepithelial migration of mucosal hemocytes in Crassostrea virginica and potential role in Perkinsus marinus pathogenesis.

We have recently described the presence of hemocytes associated with mucus covering the pallial organs (mantle, gills, and body wall) 3 of the eastern oyster Crassostrea virginica. These hemocytes, hereby designated "pallial hemocytes" share common general characteristics with circulating hemocytes but also display significant differences particularly in their cell surface epitopes. The specific location of pallial hemocytes as peripheral cells exposed directly to the marine environment confers them a putative sentinel role. The purpose of this study was to gain a better understanding of the source of these pallial hemocytes by evaluating possible exchanges between circulatory and pallial hemocyte populations and whether these exchanges are regulated by pathogen exposure. Bi-directional transepithelial migrations of hemocytes between pallial surfaces and the circulatory system were monitored using standard cell tracking approaches after staining with the vital fluorescent dye carboxyfluorescein diacetate succinimidyl ester (CFSE) in conjunction with fluorescent microscopy and flow cytometry. Results showed bi-directional migration of hemocytes between both compartments and suggest that hemocyte migration from the pallial mucus layer to the circulatory system may occur at a greater rate compared to migration from the circulatory system to the pallial mucus layer, further supporting the role of pallial hemocytes as sentinel cells. Subsequently, the effect of the obligate parasite Perkinsus marinus and the opportunistic pathogen Vibrio alginolyticus on transepithelial migration of oyster hemocytes was investigated. Results showed an increase in hemocyte migration in response to P. marinus exposure. Furthermore, P. marinus cells were acquired by pallial hemocytes before being visible in underlying tissues and the circulatory system suggesting that this parasite could use pallial hemocytes as a vehicle facilitating its access to oyster tissues. These results are discussed in light of new evidence highlighting the role of oyster pallial organs as a portal for the initiation of P. marinus infections in oysters.

Study on in vitro model of hepatitis B virus-infected transwell chambers mediated by peripheral blood mononuclear cells

Objective To observe the transport of hepatitis B virus (HBV)-infected peripheral blood mononuclear cells (PBMC) through placental barrier set up by choriocarcinoma trophoblast cells (Bewo cells), and to explore the biological role of PBMC as a carrier for HBV transport. Methods Bewo cells and PBMC were cultured and their proliferation and activity were detected by cell counting kit (CCK)-8. One hundred μL serum containing 5×106 copy/mL HBV DNA was used to infect PBMC, and cells infected with HBV were labeled by fluorescent dye carboxyfluorescein diacetate succinimidyl ester (CFSE). A co-culture model of Bewo cells and HBV-infected PBMC was set up by transwell chamber. The migration of HBV-infected PBMC was detected by flow cytometry. Realtime fluorescence quantitative polymerase chain reaction method was used to detect HBV DNA contents of PBMC under transwell chamber. Results PBMC and Bewo cells proliferated at around 24 h and entered into growth stagnation at around 120 h. The contents of PBMC labeled by green fluorescent at 0, 12, 24 and 48 h during co-culture under chamber were (0.445±0.021)%, (21.180±4.653)%, (34.830±7.156)% and (64.185±3.161)%, respectively. The amount of PBMC marked green fluorescence increased over prolonged incubation time (F=68.983, P=0.001). PBMC HBV DNA contents at 24 and 48 h of co-culture under chamber were (1.925±0.431)×103 copy/mL and (2.565±0.361)×103 copy/mL, respectively, indicating that PBMC under chamber were infected with HBV. Conclusions PBMC may be a target for HBV infection in extrahepatic tissues. Placental trophoblastic barrier built by transwell chambers may provide new ideas to investigate HBV transmission across the placenta in vitro. Key words: Hepatitis B virus; Peripheral blood mononuclear cells; Bewo cells; Transwell chamber

Cytokine-Mediated Programmed Proliferation of Virus-Specific CD8+ Memory T Cells

During infection, CD8(+) Tcells not only respond to antigenic signals through their Tcell receptor (TCR) but also incorporate inflammatory signals from cytokines produced in the local infected microenvironment. Transient TCR-mediated stimulation will result in programmed proliferation that continues despite removal of the antigenic stimulus, but it remains unclear whether brief exposure to specific cytokines will elicit similar effects. Here, we have demonstrated that brief stimulation of memory Tcells with interleukin-12 (IL-12) and interleukin-18 (IL-18) results in tightly regulated programmed proliferation, in addition to acquisition of enhanced virus-specific cytokine production and cytolytic activity. CD8(+) Tcells briefly exposed to IL-12 and IL-18 invitro showed improved antiviral activity invivo, as demonstrated by increased proliferation and reduced viremia. These results indicate that even transitory exposure to inflammatory cytokines can provide a selective advantage to infiltrating CD8(+) Tcells by triggering a developmental program that is initiated prior to direct contact with virus-infected cells.

Going deep: Single cell physiology and cell function

function as well as killing assays is of relevance in manyhigh-throughput assays where numerous different effectors,targets, or treatments have to be analyzed. In order to quanti-tate cell function modification by chemical or biologicalagents, several cell-based assays have been developed in thepast. For example, standardized polychromatic (11-color)cytokine expression assays are available to simultaneouslymeasure the intracellular production of five (1) or more cyto-kines in combination with phenotypic markers. Intracellularsignaling patterns can be investigated also, using multiplexedphosphoflow assays (2). In addition, for reliable quantitation,new multiplexed assays are in demand to reduce the numberof assays required, as well as the number of animals needed forreliable results.Inosine triphosphatase (ITPase) polymorphism has beenassociated with several diseases as well as advantageous or dis-advantageous response to therapy. In most studies ITPaseexpression was measured in erythrocyte lysates. However, itwas found that leukocytes express ITPase at substantiallyhigher levels than erythrocytes. Vroemen and coworkers fromHeerlen and Maastricht, The Netherlands (this issue, page672) investigated differences in ITPase levels in different leu-kocyte subsets of human volunteers. To this end, they devel-oped the first polychromatic flow cytometry assays to identifycell phenotypes and quantitate their ITPase expression levels.The authors found that all leukocyte subsets express ITPase.However, expression level was higher in activated monocytesand granulocytes as compared to lymphocytes. These assaysmay help in diseases where ITPase polymorphism plays a rolein order to improve our understanding of disease mechanismsand potentially produce new diagnostic tools for individua-lized therapy.The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide) (MTT) assay is a very commonly used cell-basedvitality assay for high-throughput drug screening. It is based onthe reduction of the yellow water soluble dye MTT into a blueviolet water insoluble dye, formazane (3). Now, Lim and co-workers from the Universities of Seoul and Kongju in Korea(this issue, page 691) developed a microfluidic image cytome-try-based method. Their measurements are multiplexed in thatthey do not only quantitate (on the single cell basis) the meta-bolization of MTT but in addition, also detect cell morphologi-cal changes. This assay was used to test cadmium (Cd

Use of SNARF-1 to measure murine T cell proliferation in vitro and its application in a novel regulatory T cell suppression assay

The green fluorescent dye carboxyfluorescein diacetate succinimidyl ester (CFSE) has been used to track the proliferation of T cells in vitro. Such assays often incorporate more than one population of cells, but the paucity of alternative, spectrally distinct dyes suitable for measuring proliferation has hampered the simultaneous tracking of multiple cell populations; furthermore, CFSE is not compatible with green fluorescent protein (GFP), used to identify T cells in various transgenic mice. We have therefore validated the use of the far red dye seminaphthorhodafluor-1 (SNARF)-1 – originally developed to measure intracellular pH – to track murine T cell proliferation in vitro, demonstrating its ability to distinguish multiple cycles of proliferation over three days in a similar fashion to CFSE. The small changes in fluorescence emission attributed to intracellular alkalinisation of proliferating T cells have minimal impact on the ability of SNARF-1 to track cell division and this dye induces minimal cell death at the concentration used in this application. On the basis of these results, we have developed a novel in vitro murine T cell suppression assay, in which the proliferation of both conventional T cells (Tcons) stained with SNARF-1 and regulatory T cells (Tregs) stained with CFSE can be measured simultaneously. We have also demonstrated that SNARF-1 may be used to stain Tcons in assays of suppression involving ‘designer’ Tregs, generated by the transduction of CD4+ T cells with constructs encoding the Foxp3gfp fusion protein.

An aqueous birch leaf extract of Betula pendula inhibits the growth and cell division of inflammatory lymphocytes

Leaf extracts of Betula pendula have been traditionally used for the treatment of patients with rheumatoid arthritis (RA) or osteoarthritis. We investigated the anti-proliferative capacity of an aqueous leaf extract of Betula pendula (BPE) on human primary lymphocytes in vitro, because activated lymphocytes play a major role in the initiation and maintenance of RA. Lymphocyte proliferation and cell division was measured by the activity of mitochondrial dehydrogenases and by using the membrane-permeable dye carboxyfluorescein diacetate succinimidyl ester (CFSE), respectively. Apoptosis was analyzed by surface staining of phosphatidylserine and intracellular activation of effector caspases 3 and 7 in comparison to the drug methotrexate using flow cytometric and photometrical analysis. In addition, the impact of the extract on cell cycle distribution was investigated by propidium iodide staining of DNA. For the bioassays BPE concentrations of 10-160 μg/mL were investigated. A phytochemical analysis, using LC-MS and HPLC, was conducted to identify the polyphenolic constituents of the birch leaf extract. Leaf extracts of Betula pendula inhibited the growth and cell division (CD8(+): 40 μg/mL: 45%; 80 μg/mL: 60%; 160 μg/mL: 87%) (CD4(+): 40 μg/mL: 33%; 80 μg/mL: 54%; 160 μg/mL: 79%) of activated, but not of resting T lymphocytes in a significant dose-dependent manner. The inhibition of lymphocyte proliferation due to apoptosis induction (compared to untreated control: 40 μg/mL: 163%; 80 μg/mL: 240%; 160 μg/mL: 348%) and cell cycle arrest was comparable to that of methotrexate. LC-MS analyses showed that the extract contains different quercetin-glycosides. Our results give a rational basis for the use of Betula pendula leaf extract for the treatment of immune disorders, like rheumatoid arthritis, by diminishing proliferating inflammatory lymphocytes.

Differential preferences in serosal homing and distribution of peritoneal B-cell subsets revealed by in situ CFSE labeling

Peritoneal B cells represent a heterogeneous mixture of mature peripheral B lineage subsets with distinct developmental and functional characteristics. Here, we report that a single intraperitoneal injection of intracellular fluorescent dye carboxyfluorescein diacetate succinimidyl ester (CFSE) results in the stable fluorescent labeling of resident lymphocytes, without dissipation of the tracer compound into other peripheral lymphoid organs. Using this in situ labeling procedure followed by multicolor flow cytometry or tissue fluorescence at various periods for up to 4 weeks post-labeling, we demonstrate that the distinct peritoneal leukocyte sub-populations and, within the B lineage, B-1 and B-2 B-cell subsets have different exchange kinetics with extraperitoneal sites under steady-state conditions. The B cells labeled with CFSE showed only minimal localization to other peripheral lymphoid tissues. On the other hand, a substantial fraction of both B-1 and B-2 subsets labeled with CFSE accumulated within the pleural cavity, although at a lower frequency than in the peritoneum. We also show that exposure to LPS induces a rapid re-distribution of peritoneal B lymphocytes and an enhanced entry of B-1 cells in the pleural cavity, in addition to augmenting the egress and the division-linked reduction of CFSE fluorescence of both B-1 and B-2 cells. These data indicate that following their in situ labeling, peritoneal lymphocytes show preferential accumulation in serosal cavities, although with a differential efficiencies for T, B-1 and B-2 lymphocyte subsets.

Use of the intracellular fluorescent dye CFSE to monitor lymphocyte migration and proliferation.

The stable incorporation of the intracellular fluorescent dye 5-(and -6)-carboxyfluorescein diacetate succinimidyl ester (CFSE) into cells provides a powerful tool to monitor cell migration, and to quantify cell division, because of the sequential decrease in fluorescent labeling in daughter cells. CFSE-labeled lymphocytes have been used to analyze the relationship between cell division and differentiation of cell function, and cell proliferation versus apoptosis, both in vivo and in vitro, and have allowed analysis of the site of response to antigens in vivo.

Monitoring lymphocyte proliferation in vitro and in vivo with the intracellular fluorescent dye carboxyfluorescein diacetate succinimidyl ester

This protocol outlines the carboxyfluorescein diacetate succinimidyl ester (CFSE) method for following the proliferation of human lymphocytes in vitro and mouse lymphocytes both in vitro and in vivo. The method relies on the ability of CFSE to covalently label long-lived intracellular molecules with the highly fluorescent dye, carboxyfluorescein. Following each cell division, the equal distribution of these fluorescent molecules to progeny cells results in a halving of the fluorescence of daughter cells. The CFSE labeling protocol described, which typically takes <1 h to perform, allows the detection of up to eight cell divisions before CFSE fluorescence is decreased to the background fluorescence of unlabeled cells. Protocols are outlined for labeling large and small numbers of human and mouse lymphocytes, labeling conditions being identified that minimize CFSE toxicity but maximize the number of cell divisions detected. An important feature of the technique is that division-dependent changes in the expression of cell-surface markers and intracellular proteins are easily quantified by flow cytometry.

Measuring lymphocyte proliferation, survival and differentiation using CFSE time-series data

Cellular proliferation is an essential feature of the adaptive immune response. The introduction of the division tracking dye carboxyfluorescein diacetate succinimidyl ester (CFSE) has made it possible to monitor the number of cell divisions during proliferation and to examine the relationship between proliferation and differentiation. Although qualitative examination of CFSE data may be useful, substantially more information about division and death rates can be extracted from quantitative CFSE time-series experiments. Quantitative methods can reveal in detail how lymphocyte proliferation and survival are regulated and altered by signals such as those received from co-stimulatory molecules, drugs and genetic polymorphisms. In this protocol, we present a detailed method for examining time-series data using graphical and computer-based procedures available to all experimenters.

Modeling T Cell Proliferation and Death in Vitro Based on Labeling Data: Generalizations of the Smith–Martin Cell Cycle Model

The fluorescent dye carboxyfluorescein diacetate succinimidyl ester (CFSE) classifies proliferating cell populations into groups according to the number of divisions each cell has undergone (i.e., its division class). The pulse labeling of cells with radioactive thymidine provides a means to determine the distribution of times of entry into the first cell division. We derive in analytic form the number of cells in each division class as a function of time based on the distribution of times to the first division. Choosing the distribution of time to the first division to fit thymidine labeling data for T cells stimulated in vitro under different concentrations of IL-2, we fit CFSE data to determine the dependence of T cell kinetic parameters on the concentration of IL-2. As the concentration of IL-2 increases, the average cell cycle time is shortened, the death rate of cells is decreased, and a higher fraction of cells is recruited into division. We also find that if the average cell cycle time increases with division class then the qualify of our fit to the data improves.

Quantifying division of aortal smooth muscle cells in culture stimulated by 1,25(OH)2D3

Inhibitory effect of 1α,25dihydroxycholecalciferol (1,25D3=calcitriol) in different cell type is well recognized but its promoting effect on vascular smooth muscle cells (SMCs) is poor established. Therefore, the aim of this study was to determine stimulatory effect of calcitriol on aortal SMCs proliferation in culture. We used the cell division analysis procedure based on the quantitative sequential halving of the stably incorporating fluorescent dye carboxyfluorescein diacetate succinimidyl ester (CFSE). This technique allowed the visualization of cycles of SMCs division by flow cytometry. Rat aortal SMCs were labeled with CFSE and cultured for up to 10 days with defined concentration of calcitriol in medium. Proliferative activity as the percentage of SMCs in different phases of the cell cycle using propidium iodide was determined. Apoptosis was assessed using Annexin-V/CFDA method. The results suggest that low concentrations of an active form of vitamin D—1α,25dihydroxycholecalciferol applied in supraphysiological concentration of 10nmol/l is a mitogenic factor for aortal SMCs. None of the applied concentrations of calcitriol caused apoptosis. The findings well support our morphological (LM) and ultrastructural (TEM and SEM) observations.

CD38 Expression in B-CLL Is Dynamic and Changes Due to Contact with Activating Stimuli Found within the Leukaemic Microenvironment.

High levels of CD38 expression in B-cell chronic lymphocytic leukaemia (B-CLL) confer a poor prognosis. Although its role in B-CLL is unknown, signalling through CD38 has been implicated in cell survival, trafficking and proliferation. Since proliferation in B-CLL is thought to take place within both bone marrow (BM) and secondary lymphoid tissue, we investigated whether CD38 expression might vary in response to stimuli that occur in these tissue compartments.Firstly, we compared the percentage CD38 expression of CD5/19 cells on 35 paired PB and BM aspirate B-CLL samples. The mean CD38% was significantly higher in BM than PB in all samples (27% vs 19%, p=0.009) including samples with a PB CD38 of 7% or more (33% vs 42%, p=0.047), indicating that factors present in the BM up regulate CD38 expression. Next, CD38 expression and cell division of B-CLL peripheral blood mononuclear cells (PBMCs) were examined in an in vitro system aimed at mimicking the proliferation centre microenvironment where leukaemic cells are situated in close proximity to activated T lymphocytes. Positively selected T cells from 15 B-CLL patients were activated overnight with CD3/28 beads and subsequently cultured with autologous B-CLL PBMCs. Both the percentage of CD19+ CD38+ cells (29.9% vs 59.9%, p=0.003) and CD38 mean fluorescence intensity (75.1 vs 830.8, p=0.005) increased over the 6 day culture period. B-CLL cell division was assessed using the dye carboxyfluorescein diacetate succinimidyl ester (CFSE) in the same co-culture system. This showed that co-culture with autologous activated T-cells can result in B-CLL cell division, and is preceded by CD38 up regulation. In addition, significantly more B-CLL cells underwent at least one division from patients with an initial CD38 level of 7% or more, as compared to under 7% (24.6% vs 10.9%, p=0.031). To further investigate the relationship between B-CLL cell proliferation, CD38 expression and the role of T-cells we examined tissue sections known to contain paraimmunoblasts and other proliferating B-CLL cells. Four colour confocal microscopy using CD3, Ki67, CD38 and CD23 to label frozen B-CLL lymph nodes was employed. Large Ki67+ CD23+ cells were present in close proximity to CD3+ T-cells and these large B-CLL cells had higher CD38 expression than the surrounding small B-CLL lymphocytes.These results support the proposal that CD38 expression in B-CLL is dynamic and may reflect exposure to T-cell derived stimuli which contribute to proliferation in the BM or LN microenvironment. A possible explanation for the poorer prognosis of patients with higher CD38 expression may be that their disease has more proliferative potential.

Quantifying lymphocyte kinetics in vivo using carboxyfluorescein diacetate succinimidyl ester

The cytoplasmic dye carboxyfluorescein diacetate succinimidyl ester (CFSE) is used to quantify cell kinetics. It is particularly important in studies of lymphocyte homeostasis where its labelling of cells irrespective of their stage in the cell cycle makes it preferable to deuterated glucose and BrdU, which only label dividing cells and thus produce unrepresentative results. In the past, experiments have been limited by the need to obtain a clear separation of CFSE peaks forcing scientists to adopt a strategy of in vitro labelling of cells followed by their injection into the host. Here we develop a framework for analysis of in vivo CFSE labelling data. This enables us to estimate the rate of proliferation and death of lymphocytes in situ, and thus represents a considerable advance over current procedures. We illustrate this approach using in vivo CFSE labelling of B lymphocytes in sheep.

Flow cytometric analysis of fluorescence in situ hybridization with dye dilution and DNA staining (flow‐FISH‐DDD) to determine telomere length dynamics in proliferating cells

Telomeres shorten during DNA replication; extensive erosion of telomeres likely promotes replicative senescence and chromosomal instability. Telomere length in individual cells has been quantified by flow cytometric analysis of fluorescence in situ hybridization (flow-FISH). To determine the rate of telomere attrition (telomere erosion per cell division), we combined flow-FISH with dye dilution and DNA staining (flow-FISH-DDD) and measured telomere-specific fluorescence in proliferating cells identified by cell generation and cell cycle phase. Peripheral blood mononuclear cells (PBMC) were stained with the cell division tracking dye carboxyfluorescein diacetate succinimidyl ester (CFSE), stimulated with phytohemagglutinin (PHA), grown for 5-6 days, hybridized with a telomere sequence-specific peptide nucleic acid fluorescent probe (PNA-Cy5), counterstained with DAPI, and analyzed by flow cytometry. The cell cycle distribution and cell division generations were respectively identified by analysis of DAPI emission and deconvolution of CFSE emission, and Cy5 emission was used to determine telomere-specific fluorescence, an indicator of telomere length, in each cell. In stimulated PBMC, in each cell cycle phase, the telomere-specific fluorescence diminished with increasing cell generation. The rate of decline of the telomere-specific fluorescence per cell generation did not significantly differ between cell cycle phases. Application of flow-FISH-DDD to measure mean telomere length and the rate of telomere attrition in proliferating cells may find use in studies of ageing and disease, the effects of telomere-modifying agents, and variability between individuals.

The role of natural killer T (NKT) cells in immune thrombocytopenia: is strong in vitro NKT cell activity related to the development of remission?

The role of natural killer T (NKT) cells in immune thrombocytopenia: is strong <i>in vitro</i> NKT cell activity related to the development of remission?