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Results for “Cell Tracking”

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At least 181 records · Page 10Linked to original sources

PubMedDate not supplied

Reduced irradiation pulmonary fibrosis and stromal cell migration in Smad3-/- marrow chimeric mice.

Pulmonary irradiation fibrosis involves migration to the lungs of bone marrow origin myofibroblast progenitor cells (marrow stromal cells (MSCs)). Smad3-/- mice display decreased ionizing irradiation-induced skin fibrosis, defective osteochondrogenesis and other abnormalities thought to be associated with a defective stromal cell response(s) to transforming growth factor-beta (TGFFbeta). Clonal bone marrow stromal cell lines were derived from the adherent layer of continuous bone marrow cultures of homozygous deletion recombinant negative Smad3-/- mice and Smad3+/+ littermates. Quantitation in an Automated Cell Tracking System of the in vitro single cell migratory capacity over five days demonstrated a significant decrease in locomotion in microns per 24 h of Smad3-/- compared to Smad3+/+ clonal MSC lines. Reexpression by retroviral vector transfection of the Smad3 but not control ds-red transgene restored in vitro migratory capacity. Intravenously injected GFP transgene product labeled Smad3-/- (MSCs) seeded 10-fold less effectively than ds-red transgene product labeled Smad3+/+ cells to the 80 days post 20 Gy irradiated lungs of C57BL/6J mice and proliferated less significantly for 60 days after cell injection. Female mice chimeric for male Smad3-/- compared to Smad3+/+ marrow showed decreased irradiation pulmonary fibrosis, Y+ stromal cell migration to the lungs, and improved survival. The data show that the reduced in vitro and in vivo migratory capacity of Smad3-/- bone marrow stromal cells correlates with decreased radiation pulmonary fibrosis observed in mice chimeric for Smad3-/- marrow.

Animals↗

PubMed1995

Migration of coordinated cell clusters in mesenchymal and epithelial cancer explants in vitro.

The invasion and migration occurring in primary neoplastic tissue explants were studied by using a three-dimensional collagen matrix model, subsequent time-lapse videomicroscopy, and computer-assisted cell tracking. We show that not only single cells but groups of clustered cells comprising 5 to more than 100 cells detach from the primary tumor lesion and migrate within the adjacent extracellular matrix. These clusters were highly polarized, resulting in a high directional persistence of migration. Locomoting cell clusters were observed in primary cultures from invasive oral squamous cell carcinomas (6 of 9), ductal breast carcinomas (2 of 3), and rhabdomyosarcoma (1 of 1), whereas normal oral mucosa (0 of 4) was cell cluster negative. Thus, locomoting cell clusters could be a novel and potentially important mechanism of cancer cell invasion and metastasis.

Carcinoma, Ductal, Breast↗

PubMed2006

True monolayer cell culture in a confined 3D microenvironment enables lineage informatics.

BACKGROUND: There is a need for methods to (1) track cells continuously to generate lineage trees; (2) culture cells in in vivo-like microenvironments; and (3) measure many biological parameters simultaneously and noninvasively. Herein, we present a novel imaging culture chamber that facilitates "lineage informatics," a lineage-centric approach to cytomics. METHODS: We cultured cells in a confined monolayer using a novel "gap chamber" that produces images with confocal-like qualities using standard DIC microscopy. Lineage and other cytometric data were semiautomatically extracted from image sets of neural stem and progenitor cells and analyzed using lineage informatics. RESULTS: Cells imaged in the chamber every 3 min could be tracked for at least 6 generations allowing for the construction of extensive lineage trees with multiparameter data sets at hundreds of time points for each cell. The lineage informatics approach reveals relationships between lineage, phenotype, and microenvironment. Mass transfer characteristics and 3D geometry make the chamber more in vivo-like than traditional culture systems. CONCLUSIONS: The gap chamber allows cells to be cultured, imaged, and tracked in true monolayers permitting detailed informatics analysis of cell lineage, phenotype, and fate determinants. The chamber is biomimetic and straightforward to build and use, and should find many applications in long-term cell imaging.

Animals↗

PubMed2025

DNA Methylation Analysis by Bisulfite Pyrosequencing of Mouse Embryonic Fibroblasts with Reprogramming Enhanced by Thyroid Hormones.

DNA methylation is a widely studied epigenetic mark which in mammals involves the incorporation of a methyl group to the fifth carbon of cytosines, mainly those belonging to CpG dinucleotides. It has been linked to context-dependent regulatory functions ranging from gene and repetitive DNA silencing to gene body transcriptional activity. Because of its important roles during embryonic development and cell differentiation, DNA methylation can be used to track cell reprogramming by measuring the methylation levels of pluripotency-associated factors. In this scenario, bisulfite pyrosequencing is a simple, robust, and widely used technique which allows for the quantification of DNA methylation levels at small, specific regions of the genome. It involves the amplification and biotin tagging of bisulfite-converted DNA. Single amplified strands are then purified using streptavidin and finally pyrosequenced using a sequencing primer. Thus, it is an ideal method for the quantitative profiling of specific genomic regions, with applications ranging from biomarker discovery and epigenetic clock tracking to omic validation studies.

Animals↗

PubMed1997

Influence of extremely low frequency electromagnetic fields on the swimming behavior of ciliates.

Different species of ciliates (Paramecium biaurelia, Loxodes striatus, Tetrahymena thermophila) have been taken as model systems to study the effects of extremely low-frequency electromagnetic fields (50 Hz, 0.5-2.0 mT) on the cellular level. A dose-dependent increase in the mean swimming velocity and a decrease in the linearity of cell tracks were observed in all wild-type cells. In contrast, field-exposure did not increase the number of directional turns of the Paramecium tetraurelia pawn mutant (d4-500r), which is characterized by defective Ca2+-channels. The described changes indicate a direct effect of low frequency electromagnetic fields on the transport mechanisms of the cell membrane for ions controlling the motile activity of cilia.

Animals↗

PubMed2003

Gene therapy for hematological malignancies.

Since cancer is the result of genetic mutations, it should be well suited for correction through gene therapy. Hematological malignancies in which human gene transfer has been performed are leukemias, lymphomas, graft-versus host disease after allogeneic bone marrow transplantation in leukemia, and multiple myeloma. Gene therapy may be used to induce or enhance an antitumor immunological reaction, to correct a genetic defect in the tumor cells, to render the malignant disease more susceptible to conventional therapies, to make the normal host cells more resistant to conventional therapies, or to track cells used for therapy. Gene therapy will probably be most valuable for the eradication of minimal residual disease after the use of conventional therapies.

Apoptosis↗

PubMed2003

Tor kinases are in distinct membrane-associated protein complexes in Saccharomyces cerevisiae.

Tor1p and Tor2p kinases, targets of the immune-suppressive antibiotic rapamycin, are components of a highly conserved signaling network that couples nutrient availability and cell growth. To gain insight into the molecular basis underlying Tor-dependent signaling, we used cell fractionation and immunoaffinity chromatography to examine the physical environment of Tor2p. We found that the majority of Tor2p associates with a membrane-bound compartment along with at least four other proteins, Avo1p-Avo3p and Lst8p. Using immunogold electron microscopy, we observed that Tor2p, as well as Tor1p, localizes in punctate clusters to regions adjacent to the plasma membrane and within the cell interior, often in association with characteristic membranous tracks. Cell fractionation, coimmunoprecipitation, and immunogold electron microscopy experiments confirmed that Lst8 associates with both Tor2p as well as Tor1p at these membranous sites. In contrast, we find that Kog1, the yeast homologue of the mammalian Tor regulatory protein Raptor, interacts preferentially with Tor1p. These findings provide evidence for the existence of Tor signaling complexes that contain distinct as well as overlapping components. That these complexes colocalize to a membrane-bound compartment suggests an intimate relationship between membrane-mediated signaling and Tor activity.

Animals↗

PubMed1995

Live cell image segmentation.

A major requirement of an automated, real-time, computer vision-based cell tracking system is an efficient method for segmenting cell images. The usual segmentation algorithms proposed in the literature exhibit weak performance on live unstained cell images, which can be characterized as being of low contrast, intensity-variant, and unevenly illuminated. We propose a two-stage segmentation strategy which involves: 1) extracting an approximate region containing the cell and part of the background near the cell, and 2) segmenting the cell from the background within this region. The approach effectively reduces the influence of peripheral background intensities and texture on the extraction of a cell region. The experimental results show that this approach for segmenting cell images is both fast and robust.

Algorithms↗

PubMedDate not supplied

Graviresponses of gliding and swimming Loxodes using step transition to weightlessness.

Cells of Loxodes striatus were adjusted to defined culturing, experimental solution O2-supply, temperature, and state of equilibration to be subjected to step type transition of acceleration from normal gravity, (1 g) to the weightless condition (microgravity) during free fall in a 500 m drop shaft. Cellular locomotion inside a vertical experimental chamber was recorded preceding transition and during 10 s of microgravity. Cell tracks from video records were used to separate cells gliding along a solid surface from free swimmers, and to determine gravitaxis and gravikenesis of gliding and swimming cells. With O2 concentrations > or = 40% air saturation gliders and swimmers showed a positive gravitaxis. In microgravity gravitaxis of gliders relaxed within 5 s whereas gravitaxis relaxation of swimmers was not completed even after 10 s. Rates of horizontal gliders (319 micrometers/s) exceeded those, of horizontal swimmers (275 micrometers/s). Relaxation of gravikinesis was incomplete after 10 s of microgravity. Analysis of the locomotion rates during the g-step transition revealed that gliders sediment more slowly, than swimmers (14 versus 45 micrometers/s). The gravikinesis of gliders cancelled sedimentation effects during upward and downward locomotion tending to maintain cells at a predetermined level inside sediments of a freshwater habitat. At > or = 40% air saturation, gravikinesis of swimmers augmented the speed of the majority of cells during gravitaxis, which favours fast vertical migrations of Loxodes.

Animals↗

PubMed2002

Characterization and efficacy of PKH26 as a probe to study the replication history of the human hematopoietic KG1a progenitor cell line.

The PKH26 dye can, in principle, be used for the study of asymmetric cell divisions (ASDs). A requirement for the identification of ASDs based on fluorescence intensity is that the PKH26 dye is distributed equally between daughter cells at each division, but this has not been demonstrated at a single-cell level. The efficacy of PKH26 as a probe for the study of ASDs was examined using the human hematopoietic KG1a cell. An automated time-lapse fluorescent microscope system was used to determine changes in cell size and fluorescence intensity during culture, and track cell divisions. The images of daughter cells were analyzed using the Isee software to determine the distribution of PKH26 dye between daughter cells. Ratios of cell size, mean fluorescence intensity, and total fluorescence intensity were calculated by dividing the values for one daughter cell by the value of the other daughter cell. The ratios for cell size, mean intensity, and total intensity were 1.13 +/- 0.12, 1.08 +/- 0.07, and 1.15 +/- 0.14 (mean +/- SD), respectively. Thus, PKH26 is not distributed equally to both daughter cells upon cell division. However, the replication history of individual KG1a cells can be reliably deduced for up to three divisions based solely on the mean and total fluorescence intensity of the PKH26 dye, using PKH26 concentrations below the chemical and phototoxic limits (2 microM).

Cell Division↗

PubMed2006

Increased radioresistance, g(2)/m checkpoint inhibition, and impaired migration of bone marrow stromal cell lines derived from Smad3(-/-) mice.

Smad3 protein is a prominent member of the Tgfb receptor signaling pathway. Smad3(-/-) mice display decreased radiation-induced skin fibrosis, suggesting a defect in both Tgfb-mediated fibroblast proliferation and migration. We established bone marrow stromal cell lines from Smad3(-/-) mice and homozygous littermate(+/+) mice. Smad3(-/-) cells displayed a significant increase in radiation resistance with a D(0)=2.25+/- 0.14 Gy compared to Smad3(+/+) cells with a D(0)=1.75+/- 0.03 (P=0.023). Radioresistance was abrogated by reinsertion of the human SMAD3 transgene, resulting in a D(0)=1.49 0.10 (P=0.028) for Smad3(-/-)(3) cells. More Smad3(-/-) cells than Smad3(+/+) cells were in the G(2)/M phase; Smad3(-/-)(3) cells were similar to Smad3(+/+) cells. Smad3(+/+) cells exhibited increased apoptosis 24 h after 5 Gy (15%) or 8 Gy (43%) compared to less than 1% in Smad3(-/-) cells exposed to either dose. The movement of Smad3(-/-) cells, measured in an automated cell tracking system, was slower than that of Smad3(+/+) cells. Smad3(-/-)(3) cells resembled Smad3(+/+) cells. These studies establish concordance of a defective Tgfb signal transduction pathway, an increased proportion of G(2)/M cells, and radioresistance. The decreased migratory capacity of Smad3(-/-) cells in vitro correlates with decreased radiation fibrosis in vivo in mice deficient in Tgfb signaling.

Animals↗

PubMed2005

Tracking of migrating cells under phase-contrast video microscopy with combined mean-shift processes.

In this paper, we propose a combination of mean-shift-based tracking processes to establish migrating cell trajectories through in vitro phase-contrast video microscopy. After a recapitulation on how the mean-shift algorithm permits efficient object tracking we describe the proposed extension and apply it to the in vitro cell tracking problem. In this application, the cells are unmarked (i.e., no fluorescent probe is used) and are observed under classical phase-contrast microscopy. By introducing an adaptive combination of several kernels, we address several problems such as variations in size and shape of the tracked objects (e.g., those occurring in the case of cell membrane extensions), the presence of incomplete (or noncontrasted) object boundaries, partially overlapping objects and object splitting (in the case of cell divisions or mitoses). Comparing the tracking results automatically obtained to those generated manually by a human expert, we tested the stability of the different algorithm parameters and their effects on the tracking results. We also show how the method is resistant to a decrease in image resolution and accidental defocusing (which may occur during long experiments, e.g., dozens of hours). Finally, we applied our methodology on cancer cell tracking and showed that cytochalasin-D significantly inhibits cell motility.

Adenocarcinoma↗

PubMed2003

Boswellic acid acetate induces differentiation and apoptosis in highly metastatic melanoma and fibrosarcoma cells.

The aim of the study was to investigate the antitumor and/or preventive effect of BC-4, an isomeric compound isolated from the plant Boswellia carteri Birdw. containing alpha- and beta-boswellic acid acetate in 1:1, MW 498.3. We used the MTT (3-(4,5-dimethylthiazol-2-yl) 2,5-diphenyltetrazolium bromide) assay to study the growth inhibition activity of BC-4. Tumor cells migration within a three-dimensional collagen matrix was recorded by time-lapse videomicroscopy and computer-assisted cell tracking. Topoisomerase II was isolated from mouse melanoma B16F10 cells and its activity was determined by its ability to cut plasmid pBR322 DNA. The secretion and activity of matrix metalloproteinases (MMPs) from human fibrosarcoma HT-1080 cells were determined by gelatin zymography. BC-4 was a cytostatic compound and could induce the differentiation of B16F10 mouse melanoma cells, blocked the cell population in G1 phase and inhibited topoisomerase II activity. The G1 phase population of B16F10 cells was increased from 57.4 to 87.7%, while S phase population was reduced from 33.3 to 5.9% after treatment with BC-4 at 25 microM concentration for 48 h. BC-4 also inhibited the migration activity of B16F10. BC-4 could induce apoptosis of HT-1080 cells, as proved by acridine orange fluorescence staining, Wright-Giemsa staining, electromicroscopy, DNA fragmentation and flow cytometry. BC-4 inhibited the secretion of MMPs from HT-1080 cells, too. In conclusion, if it turns out that BC-4 is a well tolerated substance, exhibiting no significant toxicity or side effects, being evaluated currently in China, BC-4 is a good candidate for the prevention of primary tumor, invasion and metastasis.

Animals↗

PubMedDate not supplied

Growing-cube isosurface extraction algorithm for medical volume data.

In medical applications, three-dimensional volume data such as CT and MRI are gathered from medical-imaging devices. Marching cube (MC) algorithm is a common routine to extract isosurfaces from volume data. The MC algorithm generates the massive number of triangles to represent an isosurface. It is difficult to render this amount of triangles in real-time on general workstations. In this paper, we present a growing-cube algorithm to reduce the number of triangles generated by the MC algorithm. Growing-cube algorithm uses a surface tracker to avoid exhaustive searching isosurfaces cell-by-cell and, therefore, it saves computation time. During surface tracking, the growing-cube algorithm adaptively merges surfaces contained in the tracked cells to reduce the number of triangles. Surfaces are merged as long as the error is within user-specified error thresholds. Therefore, the proposed algorithm can generate a variable resolution of isosurfaces according to these error parameters.

Algorithms↗

PubMedDate not supplied

Tat peptide directs enhanced clearance and hepatic permeability of magnetic nanoparticles.

Superparamagnetic nanoparticles have a number of important biomedical applications, serving as MR contrast agents for imaging specific molecular targets, as reagents for cell labeling and cell tracking, and for the isolation of specific classes of cells. We have determined the physical and biological properties of MION-47 and amino-CLIO, nanoparticles which serve as precursors for the synthesis of targeted MR contrast agents, and Tat-CLIO, a nanoparticle used as a cell labeling reagent. Blood half-lives for MION-47 and amino-CLIO were 682 +/- 34 and 655 +/- 37 min, respectively. The attachment of 9.7 tat peptides per crystal to amino-CLIO resulted in a reduction in blood half-life to 47 +/- 6 min. MION-47, amino-CLIO, and Tat-CLIO were present in highest concentrations in liver and spleen and lymph nodes, where concentrations for all three nanoparticles ranged from 8.80 to 6.11% of injected dose per gram. Twenty-four hours after the intravenous injection of amino-CLIO, the nanoparticle was concentrated in cells surrounding hepatic blood vessels (endothelial and Kupffer cells), in a fashion similar to that obtained with other nanoparticle preparations. In contrast, Tat-CLIO was present as numerous discrete foci of intense fluorescence throughout the parenchyma. Using the peptide as a component of future nanoparticles, it might be possible to design sensors for the detection of macromolecules present in intracellular compartments.

Amino Acid Sequence↗

PubMed2003

Effects of neurotransmitters on the chemokinesis and chemotaxis of MDA-MB-468 human breast carcinoma cells.

Most patients suffering from breast carcinoma do not die due to the primary tumor but from the development of metastases. Active migration of cancer cells is a prerequisite for development of these metastases. We used time-lapse videomicroscopy and computer-assisted cell tracking of MDA-MB-468 human breast carcinoma cells, which were incorporated into a three-dimensional collagen matrix, in order to analyze the migratory activity of these cells in response to different neurotransmitters. Our results show that met-enkephalin, substance P, bombesin, dopamine, and norepinephrine have a stimulatory effect on the migration of the breast cancer cells; moreover, these cells show positive chemotaxis towards norepinephrine as was analyzed by the directionality and persistence on a single-cell basis. Gamma-aminobutyric acid (GABA) however has an inhibitory effect. Endorphin and leu-enkephalin, as well as histamin and acetylcholine, had no influence on the migratory activity of the cells. In summary, we provide evidence for a strong regulatory involvement of neurotransmitters in the regulation of breast cancer cell migration, which might provide the basis for the use of the pharmacological agonists and antagonists for the chemopreventive inhibition of metastasis development.

Antineoplastic Agents↗

PubMed1999

Quantification of cellular properties from external fields and resulting induced velocity: magnetic susceptibility.

An experimental technique is discussed in which the magnetic susceptibility of immunomagnetically labeled cells can be determined on a cell-by-cell basis. This technique is based on determining the magnetically induced velocity that an immunomagnetically labeled cell has in a well-defined magnetic energy gradient. This velocity is determined through the use of video recordings of microscopic images of cells moving in the magnetic energy gradient. These video images are then computer digitized and processed using a computer algorithm, cell tracking velocimetry, which allows larger numbers (>10(3)) of cells to be analyzed.

Cells, Cultured↗

PubMed2002

Tracking transplanted stem cell migration using bifunctional, contrast agent-enhanced, magnetic resonance imaging.

The ability to track stem cell transplants in the brain by in vivo neuroimaging will undoubtedly aid our understanding of how these cells mediate functional recovery after neural transplantation. One major challenge for the development and refinement of stem cell transplantation is to map the spatial distribution and rate of migration in situ. Here we report a method for tracking transplanted stem cells in the ischemia-damaged rat hippocampus by magnetic resonance imaging (MRI). Before transplantation, stem cells were labeled in vitro either with a novel bifunctional contrast agent, gadolinium rhodamine dextran (GRID), identifiable by both MRI and fluorescence microscopy, or with PKH26, visible exclusively under fluorescence microscopy. At different time points following engraftment, the brains were evaluated by both histology and ex vivo MR imaging. Transplanted stem cells were identified by MRI only if prelabeled with GRID, whereas fluorescence microscopy detected transplanted cells using either label. The distribution of GRID-labeled stem cells identified by MRI corresponded to those detected using fluorescence microscopy. These results demonstrate that GRID-enhanced MRI can reliably identify transplanted stem cells and their migration in the brain.

Animals↗