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Results for “Cell Tracking”

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PubMed1983

Relations between parameters of step-tracking movements and single cell discharge in the globus pallidus and subthalamic nucleus of the behaving monkey.

We describe the relations between the direction, amplitude, and velocity of step-tracking arm movements and the frequency of single cell discharge in the external (GPe) and internal (GPi) segments of the globus pallidus and the subthalamic nucleus (STN) of the behaving monkey. Statistically significant relations to the direction, amplitude, and peak velocity of the movement were found in all structures studied predominantly during the movement but also during the reaction time. For movements in a particular direction, the discharge rate was frequently a linear function of the movement amplitude and/or peak velocity. The slopes of this relation differed for different cells and comprised both positive and negative values. STN differed from both GPe and GPi in that (a) a larger proportion of neurons in STN showed significant relations to the direction of movement and (b) the onset times of changes in neural activity related to movement occurred earlier in STN than in GPe or GPi. The results of these studies suggest that cells in GPe, GPi, and STN may be involved in the control of movement parameters. Loss of the basal ganglia output related to the amplitude or velocity of movement might account for the impairments of step movements observed in Parkinsonian patients. On the other hand, deranged or excessive output related to amplitude or velocity control might result in the excesses of movement observed in other disorders, such as chorea and hemiballismus. These studies also provide direct evidence that the STN exerts a specific influence on basal ganglia output related to the control of movement parameters.

Animals↗

PubMed2004

[Transferrin receptor expression of human mesenchymal stem cells and in vitro tracking by autoradiography after transplantation in spinal cord].

OBJECTIVE: To determine transferrin receptor (TfR) expression of human mesenchymal stem cells (MSCs) in vitro and after transplantation in rabbit spinal cord,and to detect implanted MSCs by in vitro autoradiography. METHODS: Human mesenchymal stem cells (hMSCs) were isolated from fetal blood. Flow cytometry assay, immuno-fluorescent staining and receptor binding assay were used to determine TfR expression of hMSCs. Radioiodinated transferrin saturated with iron [(125)I-Tf(Fe)(2)] was used as tracer. The hMSCs transplanted in rabbit spinal cord was tracked by in vitro autoradiography. Diffusion of (125)I-Tf(Fe)(2) in spinal cord was examined with autoradiography. RESULTS: TfR expression of MSCs was demonstrated by flow cytometry assay, immuno-fluorescent staining and receptor binding assay in vitro. (125)I-Tf(Fe)(2) bound to hMSCs with a equilibrium dissociation constant (KD) of (0.98+/-0.12) nmol/L and a maximal density of binding sites (B(max)) of (107 702+/-6 226) sites per cell. Immuno-fluorescent staining showed that TfRs were expressed on hMSCs on the 2nd day but not be expressed on the 10th day post transplantation. Autoradiography showed distinct accumulation of (125)I-Tf(Fe)(2) but not (125)I-HSA at hMSCs implantation sites of spinal cord sections on the 2nd day post transplantation. (125)I-Tf(Fe)(2) had diffused into spinal cord 16 hours after incubation. CONCLUSION: Implanted hMSCs could be detected by in vitro autoradiography with (125)I-Tf(Fe)(2) on the 2nd day after being transplanted in spinal cord. To track implanted hMSCs with radionuclide imaging techniques in vivo, TfR was a suitable target for imaging and radioiodinated Tf(Fe)(2) was a feasible tracer.

Animals↗

PubMed2001

Observation of antigen-dependent CD8+ T-cell/ dendritic cell interactions in vivo.

In order to track hematopoetic cells of all lineages unambiguously at all stages of development, we have developed C57BL/6 mice that express a transgene coding for green fluorescent protein (GFP) under control of the human ubiquitin C promoter. These mice, called UBI-GFP/BL6, express GFP in all tissues examined, with high levels of GFP expression observed in hematopoetic cells. UBI-GFP/BL6 mice are unique in that B cells, T cells, and dendritic cells have distinct levels of GFP fluorescence. In cell transfer experiments, leukocytes from UBI-GFP/BL6 mice are readily identified by FACS or fluorescence microscopy. We demonstrate that transplanted UBI-GFP/BL6 dendritic cells are easily identified in secondary lymphoid tissues. Direct interactions between individual dendritic cells and multiple naïve CD8+ T cells are observed in lymph nodes within 12 h of cell transfer and require loading of the dendritic cells with the appropriate peptide antigen. Dendritic cells undergo specific morphologic changes following interactions with antigen-specific T cells.

Adoptive Transfer↗

PubMed1992

Micropatterned substratum adhesiveness: a model for morphogenetic cues controlling cell behavior.

It is generally considered that tracks of cell adhesiveness are important in controlling cell migration during the development and regeneration of many tissues. In order to investigate this experimentally, a number of techniques have in the past been employed to make patterns of differential adhesiveness for in vitro studies. However, practical limitations on patterning resolution and the introduction of residual topography to the experimental substrata have restricted their usefulness. Here we describe a simplified photolithographic technique for patterning cell adhesiveness which allows a high degree of flexibility and precision. We have quantified, using adhesion and spreading characteristics of BHK cells, the differential adhesiveness that can be created on patterned surfaces, how this alters with the duration of exposure to serum proteins, and how this, in turn, relates to the persistence of cell patterning despite increases in cell density. We believe that this technique will prove extremely useful for the detailed in vitro examination of the mechanisms controlling cell behavior as it offers a degree of precision and ease of fabrication that has previously been unavailable.

Adhesiveness↗

PubMed1988

Computer-assisted analysis of the vascular endothelial cell motile response to injury.

We have developed an automated, user-friendly method to track vascular endothelial cell migration in vitro using an IBM PC/XT with MS DOS. Analog phase-contrast images of the bovine aortic endothelial cells are converted into digital images (8 bit, 250 x 240 pixel resolution) using a Tecmar Video VanGogh A/D board. Digitized images are stored at selected time points following mechanical injury in vitro. FORTRAN and assembly language subroutines have been implemented to automatically detect the wound edge and the edge of each cell nucleus in the phase-contrast, light-microscope field. Detection of the wound edge is accomplished by intensity thresholding following noise reduction in the image and subsequent sampling of the wound. After the range of wound intensities is determined, the entire image is sampled and a histogram of intensities is formed. The histogram peak corresponding to the wound intensities is subtracted, leaving a histogram peak that gives the range of intensities corresponding to the cell nuclei. Rates of cell migration, as well as cellular trajectories and cell surface areas, can be automatically quantitated and analyzed. This inexpensive, automated cell-tracking system should be widely applicable in a variety of cell biologic applications.

Animals↗

PubMed2007

Modelling the checkpoint response to telomere uncapping in budding yeast.

One of the DNA damage-response mechanisms in budding yeast is temporary cell-cycle arrest while DNA repair takes place. The DNA damage response requires the coordinated interaction between DNA repair and checkpoint pathways. Telomeres of budding yeast are capped by the Cdc13 complex. In the temperature-sensitive cdc13-1 strain, telomeres are unprotected over a specific temperature range leading to activation of the DNA damage response and subsequently cell-cycle arrest. Inactivation of cdc13-1 results in the generation of long regions of single-stranded DNA (ssDNA) and is affected by the activity of various checkpoint proteins and nucleases. This paper describes a mathematical model of how uncapped telomeres in budding yeast initiate the checkpoint pathway leading to cell-cycle arrest. The model was encoded in the Systems Biology Markup Language (SBML) and simulated using the stochastic simulation system Biology of Ageing e-Science Integration and Simulation (BASIS). Each simulation follows the time course of one mother cell keeping track of the number of cell divisions, the level of activity of each of the checkpoint proteins, the activity of nucleases and the amount of ssDNA generated. The model can be used to carry out a variety of in silico experiments in which different genes are knocked out and the results of simulation are compared to experimental data. Possible extensions to the model are also discussed.

Cell Cycle↗

PubMed2003

Carbohydrate supplementation and exercise-induced changes in T-lymphocyte function.

Carbohydrate (CHO) ingestion during exercise has been shown to reduce perturbations in immune cell numbers and function, possibly through a reduction in the cortisol response to exercise. We have previously observed that exercise decreases T-lymphocyte responses to mitogen via an increase in cell death of both CD4 and CD8 T lymphocytes (Green KJ and Rowbottom DG. J Appl Physiol. 95: 57-63, 2003). This study tested the hypothesis that CHO ingestion rather than placebo (Pl) would result in an attenuation of the cortisol response to exercise and a reduction of the exercise-associated alterations in cell death. Six well-trained cyclists completed two exercise trials consisting of 2.5 h of cycling at 85% of individual ventilatory threshold. In a random order, trials were completed under either CHO (6% CHO solution, 3.2 g CHO/kg body wt total) or Pl conditions. Blood samples were collected before exercise, midexercise (after 60 min of exercise), immediately after exercise, and after 60 min of recovery. T-lymphocyte responses to mitogen were determined by using carboxyfluorescein diacetate succinimidyl ester fluorescent cell division tracking and expansion rates, and cell death rates were calculated for each sample as well as mitosis rates for each cell generation. Cellular expansion of T lymphocytes was decreased after exercise in Pl only. The reduction in cellular expansion was related to an increase in cell death of both CD4 and CD8 cells in culture rather than a decrease in the ability of cells to undergo mitosis. CHO ingestion compared with Pl was associated with no reductions in cellular expansion or increases in cell death. CHO ingestion during exercise acted to reduce the impairment of T-lymphocyte function by decreasing cell death within mitogen-stimulated cell cultures; however, the mechanism of action appears to be independent of cortisol.

Adult↗

PubMed2006

Synectin-dependent gene expression in endothelial cells.

Synectin (GIPC1), a receptor scaffold protein, has been isolated by our laboratory as a syndecan-4 cytoplasmic domain binding partner that regulates important aspects of cell motility (Gao Y, Li M, Chen W, Simons M. J Cell Physiol 184: 373-379, 2000; Tkachenko E, Elfenbein A, Tirziu D, Simons M. Circ Res 98: 1398-1404, 2006). Moreover, synectin plays a major role in arterial morphogenesis and in growth factor signaling in arterial endothelial cells by regulating Rac1 activity (Chittenden TW, Claes F, Lanahan AA, Autiero M, Palac RT, Tkachenko EV, Elfenbein A, Ruiz de Almodovar C, Dedkov E, Tomanek R, Li W, Westmore M, Singh J, Horowitz A, Mulligan-Kehoe MJ, Moodie KL, Zhuang ZW, Carmeliet P, Simons M. Dev Cell 10: 783-795, 2006). The present study was carried out to characterize changes in synectin-dependent gene expression induced by homozygous disruption of the gene in endothelial cells. Using a combination of suppression subtraction hybridization and high throughput microarray technology, we have identified aberrant biological processes of transcriptional regulation in synectin(-/-) primary endothelial cells including abnormal basal regulation of genes associated with development, cell organization and biogenesis, intracellular tracking, and cell adhesion. Analysis of gene expression following FGF2 treatment demonstrated significant abnormalities in transcription, cytoskeletal organization and biogenesis, and protein modification and transport in synectin(-/-) compared with synectin(+/+) endothelial cells. These results confirm synectin involvement in FGF2-dependent signal transduction and provide insights into synectin-dependent gene expression in the endothelium.

Adaptor Proteins, Signal Transducing↗

PubMed2005

PEX-producing human neural stem cells inhibit tumor growth in a mouse glioma model.

A unique characteristic of neural stem cells is their capacity to track glioma cells that have migrated away from the main tumor mass into the normal brain parenchyma. PEX, a naturally occurring fragment of human metalloproteinase-2, acts as an inhibitor of glioma and endothelial cell proliferation, migration, and angiogenesis. In the present study, we evaluated the antitumor activity of PEX-producing human neural stem cells against malignant glioma. The HB1.F3 cell line (immortalized human neural stem cell) was transfected by a pTracer vector with PEX. The retention of the antiproliferative activity and migratory ability of PEX-producing HB1.F3 cells (HB1.F3-PEX) was confirmed in vitro. For the in vivo studies, DiI-labeled HB1.F3-PEX cells were stereotactically injected into established glioma tumor in nude mice. Tumor size was subsequently measured by magnetic resonance imaging and at the termination of the studies by histologic analysis including tumor volume, microvessel density, proliferation, and apoptosis rate. Histologic analysis showed that DiI-labeled HB1.F3-PEX cells migrate at the tumor boundary and cause a 90% reduction of tumor volume (P < 0.03). This reduction in tumor volume in animals treated with HB1.F3-PEX was associated with a significant decrease in angiogenesis (44.8%, P < 0.03) and proliferation (23.6%, P < 0.03). These results support the use of neural stem cells as delivery vehicle for targeting therapeutic genes against human glioma.

Animals↗

PubMed2003

Kindling fluorescent proteins for precise in vivo photolabeling.

Photobleaching of green fluorescent protein (GFP) is a widely used approach for tracking the movement of subcellular structures and intracellular proteins. Although photobleaching is a powerful technique, it does not allow direct tracking of an object's movement and velocity within a living cell. Direct tracking becomes possible only with the introduction of a photoactivated fluorescent marker. A number of previous studies have reported optically induced changes in the emission spectra of fluorescent proteins. However, the ideal photoactivated fluorescent marker should be a nonfluorescent tag capable of "switching on" (i.e., becoming fluorescent) in response to irradiation by light of a particular wavelength, intensity, and duration. In this report, we generated a mutant of Anemonia sulcata chromoprotein asCP. The mutant protein is capable of unique irreversible photoconversion from the nonfluorescent to a stable bright-red fluorescent form ("kindling"). This "kindling fluorescent protein" (KFP1) can be used for precise in vivo photolabeling to track the movements of cells, organelles, and proteins. We used KFP1 for in vivo cell labeling in mRNA microinjection assays to monitor Xenopus laevis embryo development and to track mitochondrial movement in mammalian cells.

Animals↗

PubMed1979

Target velocity signals of visual tracking in vermal Purkinje cells of the monkey.

Discharges of Purkinje cells were recorded from the vermis, lobules VI and VII, of a monkey trained to track a visual target. When the monkey tracked a sinusoidally oscillating target, cellular activity changes in phase with the velocity signal of the eye movement. When the monkey fixated a stationary point, almost identical modulation in activity occurred, reflecting the velocity signal of the motion of the retinal image of the target. The data suggest that the vermis participates in the control of smooth pursuit eye movements by providing the oculomotor system with the actual target velocity information which is the sum of eye velocity and retinal image velocity signals.

Action Potentials↗

PubMed1986

Seeding of malignant cells into the needle track after lung and pleural biopsy.

Transthoracic needle biopsy is frequently performed for the diagnosis of pulmonary and pleural disease. Seeding of malignant cells is a potential complication but is extremely uncommon. We report the second patient with malignant seeding after fine-needle aspiration biopsy of a bronchogenic carcinoma. We also report a patient with seeding of bronchogenic carcinoma after a pleural biopsy. Because seeding is a rare complication, it does not affect the indications for biopsy.

Adenocarcinoma, Papillary↗

PubMed2004

Monitoring cell therapy using iron oxide MR contrast agents.

Given the remarkable progress that has recently been obtained in animal studies, the clinical use of stem and progenitor cells to correct or replace defective cell populations may soon become a reality. In order to develop effective cell therapies, the location and distribution of these cells must be determined in a non-invasive manner. Magnetic resonance (MR) tracking of magnetically labeled cells following transplantation or transfusion may fulfill this requirement. Indeed, a series of recent studies indicate that MRI cell tracking has great potential for further evaluation and optimization of cell therapy. Due to its biocompatibility and strong effects on T2(*) relaxation, iron oxide nanoparticles appear to be the contrast agent of choice, and several methods now exist to shuttle sufficient amount of these compounds into cells. Most of the tracking work has been carried out in disease models of the central nervous system, but, recently, the infarcted heart has also received attention. With its excellent spatial resolution and the ability to track labeled cells over prolonged periods of time, MR monitoring of cell therapy is likely to become an important technique in the foreseeable future.

Animals↗

PubMed2006

A fusion tag enabling optical marking and tracking of proteins and cells by FRET-acceptor photobleaching.

Combined time-lapse imaging with optical marking of fluorescent proteins (FPs) is a widely used method in studies of the dynamic behaviour of proteins, organelles and cell populations. Most of the approaches have specific limitations as they do not permit simultaneous observation of marked and non-marked molecules, require co-expression of two FP-tagged proteins or rely on oligomerizing FPs. Here we provide a strategy to overcome such limitations with a fluorescence resonance energy transfer-competent tandem fusion tag composed of two FPs. We combine optical marking by acceptor photobleaching with spectral imaging to discriminate between marked and non-marked molecules. Such 'bleach-labelling' may be employed in a broad range of studies for robust real-time tracking of proteins, organelles and cells.

Amino Acid Sequence↗

PubMed2004

Noninvasive monitoring and tracking of muscle stem cell transplants.

BACKGROUND: Efficient techniques to noninvasively monitor stem cell transplants will accelerate the development of stem cell therapies. Magnetic resonance (MR) imaging of labeled stem cells is a noninvasive approach that can provide images with high spatial resolution. In this report, we have evaluated the ability of a commercially available, FDA-approved contrast agent to allow for the monitoring of therapeutic stem cell transplants in murine dystrophic muscle. METHODS: Multipotent, muscle-derived stem cells were labeled by incubation with ferumoxide:polycation complexes. Labeled stem cells were evaluated for the ability to differentiate into mature myotubes and be detected by high resolution MR images in vitro and in vivo. RESULTS: Endosomal accumulation of superparamagnetic iron-oxide resulted in changes in the MR contrast T(2) and T(2)*, allowing for three dimensional, noninvasive detection of labeled cells. Relaxivity measurements on cell phantoms indicate that less than eight labeled cells could be detected by MR imaging. Furthermore, therapeutic cellular grafts transplanted into dystrophic muscle could be imaged sequentially and these noninvasive images correlated with histological images obtained by conventional microscopy. Additional studies revealed that MR imaging is applicable to tracking the migration of a small number of labeled cells following arterial delivery. CONCLUSIONS: MR monitoring is a highly sensitive technique that is applicable to muscle stem cell transplantation. We anticipate that it will enhance stem cell investigations by reducing the need for invasive tissue harvests and biopsies.

Animals↗

PubMed2002

In vivo trafficking, cell cycle activity, and engraftment potential of phenotypically defined primitive hematopoietic cells after transplantation into irradiated or nonirradiated recipients.

Recent interest in bone marrow (BM) transplantation in nonconditioned or minimally conditioned recipients warrants investigation of homing patterns of transplanted hematopoietic progenitor cells (HPCs) in irradiated and nonirradiated recipients. To this end, phenotypically defined populations of BM cells were tracked in lethally irradiated or nonirradiated mice at 1, 3, 6, and 24 hours after transplantation. Recovery of transplanted cells at all time points was higher in BM of nonirradiated mice, similar to earlier suggestions. The percentage of lineage-negative Sca-1(+) cells and Sca-1(+) cells expressing CD43, CD49e, and CD49d steadily increased in BM of nonirradiated mice up to 24 hours, while fluctuating in irradiated mice. Cell cycle status and BrdU incorporation revealed that less than 20% of Sca-1(+) cells and fewer Sca-1(+)lin(-) cells had cycled by 24 hours after transplantation. To more directly examine trafficking of primitive HPCs, purified grafts of CD62L(-) or CD49e(+) subfractions of Sca-1(+)lin(-) cells, previously shown to be enriched for long-term repopulating cells, also were tracked in vivo. Recovery of purified cells was similarly increased in BM of nonirradiated mice. When 50 to 100 of these BM-homed cells were examined in serial transplantation studies, BM-homed cells from initially nonirradiated mice were enriched 5- to 30-fold for cells capable of long-term hematopoiesis in secondary recipients. Collectively, these data suggest that homing or survival of transplanted cells in irradiated recipients is less efficient than that in nonirradiated recipients, implicating an active role of radiation-sensitive microenvironmental cues in the homing process. These results may have important clinical implications in the design of BM transplantation protocols.

Animals↗

PubMed2005

An automated method for analysis of flow characteristics of circulating particles from in vivo video microscopy.

The behavior of white and red blood cells, platelets, and circulating injected particles is one of the most studied areas of physiology. Most methods used to analyze the circulatory patterns of cells are time consuming. We describe a system named CellTrack, designed for fully automated tracking of circulating cells and micro-particles and retrieval of their behavioral characteristics. The task of automated blood cell tracking in vessels from in vivo video is particularly challenging because of the blood cells' nonrigid shapes, the instability inherent in in vivo videos, the abundance of moving objects and their frequent superposition. To tackle this, the CellTrack system operates on two levels: first, a global processing module extracts vessel borders and center lines based on color and temporal patterns. This enables the computation of the approximate direction of the blood flow in each vessel. Second, a local processing module extracts the locations and velocities of circulating cells. This is performed by artificial neural network classifiers that are designed to detect specific types of blood cells and micro-particles. The motion correspondence problem is then resolved by a novel algorithm that incorporates both the local and the global information. The system has been tested on a series of in vivo color video recordings of rat mesentery. Our results show that the synergy between the global and local information enables CellTrack to overcome many of the difficulties inherent in tracking methods that rely solely on local information. A comparison was made between manual measurements and the automatically extracted measurements of leukocytes and fluorescent microspheres circulatory velocities. This comparison revealed an accuracy of 97%. CellTrack also enabled a much larger volume of sampling in a fraction of time compared to the manual measurements. All these results suggest that our method can in fact constitute a reliable replacement for manual extraction of blood flow characteristics from in vivo videos.

Algorithms↗

PubMed2000

Mechanics of living cells measured by laser tracking microrheology.

To establish laser-tracking microrheology (LTM) as a new technique for quantifying cytoskeletal mechanics, we measure viscoelastic moduli with wide bandwidth (5 decades) within living cells. With the first subcellular measurements of viscoelastic phase angles, LTM provides estimates of solid versus liquid behavior at different frequencies. In LTM, the viscoelastic shear moduli are inferred from the Brownian motion of particles embedded in the cytoskeletal network. Custom laser optoelectronics provide sub-nanometer and near-microsecond resolution of particle trajectories. The kidney epithelial cell line, COS7, has numerous spherical lipid-storage granules that are ideal probes for noninvasive LTM. Although most granules are percolating through perinuclear spaces, a subset of perinuclear granules is embedded in dense viscoelastic cytoplasm. Over all time scales embedded particles exhibit subdiffusive behavior and are not merely tethered by molecular motors. At low frequencies, lamellar regions (820 +/- 520 dyne/cm(2)) are more rigid than viscoelastic perinuclear regions (330 +/- 250 dyne/cm(2), p < 0.0001), but spectra converge at high frequencies. Although the actin-disrupting agent, latrunculin A, softens and liquefies lamellae, physiological levels of F-actin, alone (11 +/- 1.2 dyne/cm(2)) are approximately 70-fold softer than lamellae. Therefore, F-actin is necessary for lamellae mechanics, but not sufficient. Furthermore, in time-lapse of apparently quiescent cells, individual lamellar granules can show approximately 4-fold changes in moduli that last >10 s. Over a broad range of frequencies (0.1-30, 000 rad/s), LTM provides a unique ability to noninvasively quantify dynamic, local changes in cell viscoelasticity.

Actins↗