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Results for “Cell Tracking”

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PubMed1999

Fluorescent dyes for lymphocyte migration and proliferation studies.

Fluorescent dyes are increasingly being exploited to track lymphocyte migration and proliferation. The present paper reviews the properties and performance of some 14 different fluorescent dyes that have been used during the last 20 years to monitor lymphocyte migration. Of the 14 dyes discussed, two stand out as being the most versatile in terms of long-term tracking of lymphocytes and their ability to quantify lymphocyte proliferation. They are the intracellular covalent coupling dye carboxyfluorescein diacetate succinimidyl ester (CFSE) and the membrane inserting dye PKH26. Both dyes have the advantage that they can be used to track cell division, both in vitro and in vivo, due to the progressive halving of the fluorescence intensity of the dyes in cells after each division. However, CFSE appears to have the edge over PKH26 based on homogeneity of lymphocyte staining and cost. Two other fluorescent dyes, although not suitable for lymphocyte proliferation studies, are valuable tracking dyes for short-term (up to 3 day) lymphocyte migration experiments, namely the DNA-binding dye Hoechst 33342 and the cytoplasmic dye calcein. In the future it is highly likely that additional fluorescent dyes, with different spectral properties to CFSE, will become available, as well as membrane inserting fluorescent dyes that more homogeneously label lymphocytes than PKH26.

Animals↗

PubMed2005

[Labeling of mesenchymal stem cells with different superparamagnetic particles of iron oxide and detectability with MRI at 3T].

PURPOSE: In vitro evaluation of labeling efficiency of human mesenchymal stem cells (hMSCs) with different types of superparamagnetic iron oxide nanoparticles as well as detection and quantification by MRI at 3T. MATERIAL AND METHODS: hMSCs were incubated for 24 hours with 5 ultrasmall superparamagnetic particles of iron oxide (USPIO) contrast agents (1 : 30 - 1 : 30,000) of different size, coating and core compound: Endorem, Resovist, citric acid coated magnetite cores of 3 nm (CMF3), 7 nm (CMF7) and 12 nm (CoF, core: cobalt ferrite). Iron uptake, intracellular retention, detection and quantification were evaluated with MRI up to 5 weeks after incubation by cytological analysis (Prussian blue), atomic absorption spectrometry and MR relaxometry measurements. RESULTS: An effective labeling of hMSCs was achieved using Resovist, CMF3 and CMF7 with mean iron concentrations of 5.1/1.8, 1.9/1.4 and 1.5/1.0 pg/cell (dilutions 1:30 [933, 2100, 2800 microg Fe/ml]/1 : 300 [93, 210, 280 microg Fe/ml]) compared with 0.58/0.34 and 0.43/0.30 pg/cell (Endorem, CoF, dilution 1 : 30 [400, 4200 microg Fe/ml]/1 : 300 [40, 420 microg Fe/ml] unlabelled control cells: 0.01 pg/cell). Particle uptake correlated with the concentration of USPIO in the incubation medium. Detection of 5 x 10 (4) labelled cells/ml with MRI was possible up to 5 weeks after incubation (Resovist, CMF7 and CMF3). MR relaxometry measurements showed a strong correlation between cellular iron load and R2* (1/T2*), r > 0.78. No changes in cell viability or toxic effects were found. CONCLUSION: Efficiency of labeling hMSCs with USPIOs depends on coating, size and core compound of used particles. Carboxydextran-coated, clinically approved SPIO (Resovist, 50 nm) or ultrasmall citrate-coated particles (< 10 nm) result in an improved cellular uptake. In principle, the long intracellular retention of particles offers the possibility of cell tracking and migration monitoring in MRI.

Cells, Cultured↗

PubMed2001

[Correlation between the biological behavior and invasion potential in three mouse melanoma cell lines].

OBJECTIVE: To study the correlation between biological behavior and invasion potential in three homologous mouse melanoma cell lines (B16, B16F10, B16BL6). METHODS: The invasion potential of melanoma cells was assayed in a Transwell cell culture chamber. Melanin content was determined by the A value (optical density) at 470 nm in spectrophotometry. Tumor cells migration within the 3-D collagen matrix was microscopically recorded with a time-lapse video recorder and computer-assisted cell tracking system. Gelatin zymography was adopted to assay the type IV collagenase secretion. A PCR-based telomeric repeat amplification protocol (TRAP) was used for assaying telomerase activity. RESULTS: B16BL6 and B16F10 cells showed higher invasion potential than B16, but the melanin content in B16F10 was very low. B16F10 and B16BL6 possessed a higher migration ability, secreting much more type IV collagenase than the B16 though without significant difference as discovered in telomerase activity of the three melanomas cell lines. CONCLUSION: Invasion capability of mouse melanoma is closely correlated to tumor cells migration ability and secretion of type IV collagenase. Melanin content and telomerase activity are not related to the invasion ability of mouse melanoma cells.

Animals↗

PubMed2001

Migration and metalloproteinases determine the invasive potential of mouse melanoma cells, but not melanin and telomerase.

The biological characteristic cell locomotion and invasion, melanin content, metalloproteinases and telomerase activity were studied in a parental mouse melanoma cell line B16 and two descendents B16BL6 and B16F10. The invasive potential of melanoma cells was assayed in a transwell cell culture chamber. Melanin content was determined by the absorbance value at 470 nm per 10(6) cells. Tumor cells migration within the 3-D collagen matrix was microscopically recorded with a time-lapse video recorder and analyzed by computer-assisted cell tracking. Gelatin zymography was adopted to assay the metalloproteinases secretion. A polymerase chain reaction-based telomeric repeat amplification protocol (TRAP) was used for measuring telomerase activity. The results demonstrated that B16BL6 and B16F10 cells were highly invasive compared to B16 cells, but the melanin content of B16F10 was very low. B16F10 and B16BL6 were hypermotile and secreted much more metalloproteinases than B16. No differences were observed in telomerase activity among the three melanoma cell lines. Invasion of mouse melanoma was closely correlated to tumor cell migration and secretion of metalloproteinases. Melanin content and telomerase activity were phenotypically not related to invasiveness in these three mouse melanoma cell lines.

Animals↗

PubMed2006

MAGfect: a novel liposome formulation for MRI labelling and visualization of cells.

Cellular entry of imaging probes, such as contrast agents for magnetic resonance imaging (MRI), is a key requirement for many molecular imaging studies, particularly imaging intracellular events and cell tracking. Here, we describe the successful development and in vitro analysis of MAGfect, a novel liposome formulation containing a lipidic gadolinium contrast agent for MRI, Gd-DOTA-Chol , designed to enter and label cells. Liposome formulation and cell incubation time were optimised for maximum cellular uptake of the imaging probe in a variety of cell lines. MRI analysis of cells incubated with MAGfect showed them to be highly MRI active. This formulation was examined further for cytotoxicity, cell viability and mechanism of cell labelling. One of the key advantages of using MAGfect as a labelling vehicle arises from its potential for additional functions, such as concomitant drug or gene delivery and fluorescent labelling. The gadolinium liposome was found to be an effective vehicle for transport of plasmid DNA (pDNA) into cells and expression levels were comparable to the commercial transfection agent Trojene.

Cells↗

PubMed2006

In vivo magnetic resonance imaging of single cells in mouse brain with optical validation.

In the current work we demonstrate, for the first time, that single cells can be detected in mouse brain in vivo using magnetic resonance imaging (MRI). Cells were labeled with superparamagnetic iron oxide nanoparticles and injected into the circulation of mice. Individual cells trapped within the microcirculation of the brain could be visualized with high-resolution MRI using optimized MR hardware and the fast imaging employing steady state acquisition (FIESTA) pulse sequence on a 1.5 T clinical MRI scanner. Single cells appear as discrete signal voids on MR images. Direct optical validation was provided by coregistering signal voids on MRI with single cells visualized using high-resolution confocal microscopy. This work demonstrates the sensitivity of MRI for detecting single cells in small animals for a wide range of application from stem cell to cancer cell tracking.

Animals↗

PubMedDate not supplied

Macrocyclic chelators with paramagnetic cations are internalized into mammalian cells via a HIV-tat derived membrane translocation peptide.

A major obstacle to using paramagnetic MR contrast agents for in vivo cell tracking or molecular sensing is their generally low cellular uptake. In this study, we show that a paramagnetically labeled DOTA chelator derivatized with a 13-mer HIV-tat peptide is efficiently internalized into mammalian cells. Intracellular concentrations were attained that were readily detectable by MR imaging using both gadolinium and dysprosium chelates. Using this paradigm, it should be feasible to internalize a variety of chemically different agents into mammalian cells.

Amino Acid Sequence↗

PubMed2003

Temporal variations in cell migration and traction during fibroblast-mediated gel compaction.

Current models used in our laboratory to assess the migration and traction of a population of cells within biopolymer gels are extended to investigate temporal changes in these parameters during compaction of mechanically constrained gels. The random cell migration coefficient, micro (t) is calculated using a windowing technique by regressing the mean-squared displacement of cells tracked at high magnification in three dimensions with a generalized least squares algorithm for a subset of experimental time intervals, and then shifting the window interval-by-interval until all time points are analyzed. The cell traction parameter, tau(0)(t), is determined by optimizing the solution of our anisotropic biphasic theory to tissue equivalent compaction. The windowing technique captured simulated sinusoidal and step changes in cell migration superposed on a persistent random walk in simulated cell movement. The optimization software captured simulated time dependence of compaction on cell spreading. Employment of these techniques on experimental data using rat dermal fibroblasts (RDFs) and human foreskin fibroblasts (HFFs) demonstrated that these cells exhibit different migration-traction relationships. Rat dermal fibroblast migration was negatively correlated to traction, suggesting migration was not the driving force for compaction with these cells, whereas human foreskin fibroblast migration was positively correlated to traction.

Algorithms↗

PubMed2004

v-Src accelerates spontaneous motility via phosphoinositide 3-kinase, phospholipase C and phospholipase D, but abrogates chemotaxis in Rat-1 and MDCK cells.

In Rat-1 fibroblasts, v-Src causes a profound remodelling of cortical actin cytoskeleton. This transformation includes membrane ruffling, a hallmark of the leading edge in migrating cells, and results from activation of phosphoinositide 3-kinase (PI 3-kinase), phospholipase C (PLC) and phospholipase D (PLD). We therefore reexamined whether motility is constitutively triggered by v-Src and studied whether this response is controlled by the same signalling pathway. The study was performed using Rat-1/tsLA29 and MDCK/tsLA31 cells, each harbouring a different thermosensitive v-Src kinase, active at 34 degrees C but inactivated at 40 degrees C. In both cell lines, overnight v-Src activation induced transformation and accelerated spontaneous motility by approximately twofold, as evidenced by wound-healing assay and by single-cell track, time-lapse recording in Dunn chambers. Inhibitors of PI 3-kinase, PLC and PLD selectively abrogated acceleration of motility by v-Src. Since mechanisms that co-ordinate spontaneous, as distinct from oriented, cell migration are separable, we further analysed in Dunn chambers chemotactic response of Rat-1/tsLA29 cells to PDGF and of MDCK/tsLA31 cells to EGF. In both cases, v-Src decreased the steady-state level of growth factor receptors at the cell surface twofold, and abrogated movement directionality at comparable level of occupancy as in non-transformed cells. The burst of pinocytosis in response to growth factors was also abolished by v-Src. Altogether, these results indicate that v-Src triggers motility in a PI 3-kinase-, PLC- and PLD-dependent manner, but abrogates directionality by suppressing polarised signalling downstream of growth factor receptors.

Actins↗

PubMed2005

Molecular optical imaging: applications leading to the development of present day therapeutics.

A number of advances in the molecular imaging field have led to the sensing of specific molecular targets and pathways in living animals. In the optical imaging field, these include the designing of biocompatible near-infrared fluorochromes, development of targeted and activatable "smart" imaging probes, and engineering of activatable fluorescent and bioluminescent proteins. The current advances in molecular optical imaging will help in early disease diagnoses, functioning of a number of pathways and finally help speed drug discovery. In this review, we will describe the near infrared fluorescent and bioluminescence imaging modalities and how these techniques have been employed in current research. Furthermore, we will also shed some light on the use of these imaging modalities in neurotherapeutics, for example imaging different parameters of vector-mediated gene expression in glioma tumors and stem cell tracking in vivo.

Animals↗

PubMed2002

Segmentation and tracking of migrating cells in videomicroscopy with parametric active contours: a tool for cell-based drug testing.

This paper presents a segmentation and tracking method for quantitative analysis of cell dynamics from in vitro videomicroscopy data. The method is based on parametric active contours and includes several adaptations that address important difficulties of cellular imaging, particularly the presence of low-contrast boundary deformations known as pseudopods, and the occurence of multiple contacts between cells. First, we use an edge map based on the average intensity dispersion that takes advantage of relative background homogeneity to facilitate the detection of both pseudopods and interfaces between adjacent cells. Second, we introduce a repulsive interaction between contours that allows correct segmentation of objects in contact and overcomes the shortcomings of previously reported techniques to enforce contour separation. Our tracking technique was validated on a realistic data set by comparison with a manually defined ground-truth and was successfully applied to study the motility of amoebae in a biological research project.

Algorithms↗

PubMed2003

Red blood cell magnetophoresis.

The existence of unpaired electrons in the four heme groups of deoxy and methemoglobin (metHb) gives these species paramagnetic properties as contrasted to the diamagnetic character of oxyhemoglobin. Based on the measured magnetic moments of hemoglobin and its compounds, and on the relatively high hemoglobin concentration of human erythrocytes, we hypothesized that differential migration of these cells was possible if exposed to a high magnetic field. With the development of a new technology, cell tracking velocimetry, we were able to measure the migration velocity of deoxygenated and metHb-containing erythrocytes, exposed to a mean magnetic field of 1.40 T and a mean gradient of 0.131 T/mm, in a process we call cell magnetophoresis. Our results show a similar magnetophoretic mobility of 3.86 x 10(-6) mm(3) s/kg for erythrocytes with 100% deoxygenated hemoglobin and 3.66 x 10(-6) mm(3) s/kg for erythrocytes containing 100% metHb. Oxygenated erythrocytes had a magnetophoretic mobility of from -0.2 x 10(-6) mm(3) s/kg to +0.30 x 10(-6) mm(3) s/kg, indicating a significant diamagnetic component relative to the suspension medium, in agreement with previous studies on the hemoglobin magnetic susceptibility. Magnetophoresis may open up an approach to characterize and separate cells for biochemical analysis based on intrinsic and extrinsic magnetic properties of biological macromolecules.

Cell Movement↗

PubMed2004

Capacity of human monocytes to phagocytose approved iron oxide MR contrast agents in vitro.

To evaluate the capacity of human monocytes to phagocytose various approved iron oxide based magnetic resonance (MR) contrast agents and to optimize in vitro labeling of these cells. Human monocytes were incubated with two superparamagnetic iron oxide particles (SPIO) as well as two ultrasmall SPIO (USPIO) at varying iron oxide concentrations and incubation times. Iron uptake in monocytes was proven by histology, quantified by atomic emission absorption spectrometry and depicted with T2* weighted fast field echo (FFE) MR images at 1.5 T. Additionally, induction of apoptosis in iron oxide labeled monocytes was determined by YO-PRO-1 staining. Cellular iron uptake was significantly (P<0.01) higher after incubation with SPIO compared with USPIO. For SPIO, the iron oxide uptake was significantly (P<0.01) higher after incubation with the ionic Ferucarbotran as compared with the non-ionic Ferumoxides. Efficient cell labeling was achieved after incubation with Ferucarbotran at concentrations > or = 500 microg Fe/ml and incubation times > or = 1 h, resulting in a maximal iron oxide uptake of up to 50 pg Fe/cell without impairment of cell viability. In vitro labeling of human monocytes for MR imaging is most effectively obtained with the approved SPIO Ferucarbotran. Potential subsequent in vivo cell tracking applications comprise, e.g. specific targeting of inflammatory processes.

Apoptosis↗

PubMed2000

Magnetophoretic mobilities correlate to antibody binding capacities.

METHODS: A methodology and a mathematical theory have been developed, which allow quantitation of the expression levels of cellular surface antigens using immunomagnetic labels and cell tracking velocimetry (CTV) technology. RESULTS: Quantum Simply Cellular (QSC) microbeads were immunomagnetically labeled with anti-CD2 fluorescein isothiocyanate (FITC) antibodies and anti-FITC MACS paramagnetic nanoparticles. Magnetophoretic mobility has been defined as the magnetically induced velocity of the labeled cell or microbead divided by the magnetophoretic driving force, proportional to the magnetic energy density gradient. DISCUSSION: Using computer imaging and processing technology, the mobility measurements were accomplished by microscopically recording and calculating the velocity of immunomagnetically labeled QSC microbeads in a nearly constant magnetic energy gradient. A calibration curve correlating the measured magnetophoretic mobility of the immunomagnetically labeled microbeads to their antibody binding capacities (ABC) has been obtained. CONCLUSION: The results, in agreement with theory, indicate a linear relationship between magnetophoretic mobility and ABC for microbeads with less than 30,000 ABC. The mathematical relationships and QSC standardization curve obtained allow determination of the number of surface antigens on similarly immunomagnetically labeled cells.

Antigens, Surface↗

PubMed2001

T-cell apoptosis induced by granulocyte colony-stimulating factor is associated with retinoblastoma protein phosphorylation and reduced expression of cyclin-dependent kinase inhibitors.

Peripheral blood progenitor cells (PBPC) mobilized by granulocyte colony-stimulating factor (G-CSF) promptly engraft allogeneic recipients after myeloablative chemotherapy for hematologic malignancies. Surprisingly, no exacerbation of acute graft-vs-host disease has been observed despite a 10-fold higher T-cell content in PBPC compared with bone marrow allografts. Because G-CSF can suppress T-cell proliferation in response to mitogens and enhance their activation-induced apoptosis, we examined the molecular mechanisms underlying G-CSF-induced immune dysfunction. Normal allogeneic lymphocytes were challenged with phytohemagglutinin in the presence of serum collected after G-CSF administration (postG) to healthy PBPC donors, and the expression of key components of the cell cycle and apoptotic machineries was investigated by flow cytometry and Western blotting. Lymphocyte stimulation was associated with collapse of mitochondrial transmembrane potential, hypergeneration of reactive oxygen intermediates, and activation of caspase-3 and DNA fragmentation. Lymphocytes were arrested in a G(1)-like phase of the cell cycle, as measured by G(1)-phase cyclin expression and bromodeoxyuridine (BrdUrd) incorporation. Cell tracking experiments confirmed the occurrence of a lower number of population doublings in postG compared with preG cultures. Unexpectedly, the phosphorylation state of the protein encoded by the retinoblastoma susceptibility gene (pRB) was unaltered in postG cultures, and the inhibition of cell cycle progression occurred without the recruitment of the cyclin-dependent kinase inhibitors p15(INK4B), p16(INK4A), and p27(Kip1). We eventually evaluated the ability of antioxidant/cytoprotectant agents to prevent the G-CSF-induced mitochondrial dysfunction and inhibition of cell cycle progression. Of interest, both N-acetylcysteine and amifostine reduced apoptotic cell death by 45% on average, inhibited the activation/processing of caspase-3, and increased BrdUrd incorporation in postG cultures. Based on these experimental findings, a model is proposed in which T-cell activation in the presence of serum immunoregulatory factor(s) induced by G-CSF is associated with a molecular phenotype mimicking the G(1)-S transition and consisting of pRB phosphorylation, lack of CDKI recruitment, and reduced cyclin-E expression. The putative relationship between lymphocyte mitogenic unresponsiveness and apoptosis induction would occur at the level of key molecules shared by the cell cycle and apoptotic machineries. Whether the G-CSF-mediated modulation of lymphocyte functions in vitro is beneficial in transplantation medicine remains to be determined.

Acetylcysteine↗

PubMed1995

Dynamic functional and structural analysis of living cells: new tools for vital staining of nuclear DNA and for characterisation of cell motion.

Increasing interest has been paid to applications of fluorescence measurements to analyze physiological mechanisms in living cells. However, few studies have taken advantage of DNA quantification by fluorometry for dynamic assessment of chromatin organization as well as cell motion during the cell cycle. This approach involves both optimal conditions for DNA staining and cell tracking methods. In this context, this report describes a stoichiometric method for nuclear DNA specific staining, using the bisbenzimidazole dye Hoechst 33342 associated with verapamil, a calcium membrane channel blocker. This method makes it possible to correlate variations of nuclear DNA content with cell motion in cells that are maintained alive. Motion measurement is the second goal of this paper and it explains the snake-spline method, and the associated cell following method.

Animals↗

PubMed2003

Anti-invasion and anti-angiogenesis effect of taxol and camptothecin on melanoma cells.

Two highly invasive melanoma cell lines B16BL6 and B16F10 were used to investigate the anti-invasion and antiangiogenesis action of taxol and camptothecin (CPT). The adhesion of melanoma cells was tested by optical absorbance at 545 nm. The invasive activity of these cells was tested in a transwell chamber assay. The cell migration within a 3D collagen matrix was recorded with a time-lapse video recorder and analyzed by computer-assisted cell tracking. Gelatin zymography was used to study the metalloproteinase activity. The chicken chorioallantoic membrane (CAM) model was used to study the anti-angiogenesis effect of the two drugs. The results demonstrated that both taxol and CPT could inhibit the migration of B16F10 cells, and inhibit the adhesion of B16F10 to fibronectin and laminin. They can reduce the metalloproteinase secretion of HT1080 and exhibit the antiangiogenesis effect in the CAM model. Taxol showed a highly anti-invasion effect on B16BL6 cells while CPT did not exhibit such an effect.

Animals↗

PubMedDate not supplied

Red cell injury assessed in a numeric model of a peripheral dialysis needle.

The highest shear stresses in a dialysis system are expected to be found in the needle, where the largest velocity-diameter ratio appears. Shear is a known source of hemolysis and related patients' discomfort. To assess the magnitude of blood cell injury and the location of its sources, a finite element model is used to calculate three-dimensional velocities and shear stresses in peripheral dialysis needles, concentrically placed in a rigid wall fistula. The boundary conditions consist of time dependent in vivo measured pressures. Cell damage is computed for different cell tracks into the needle by means of Wurzinger's empirical formula, which expresses the hemoglobin (Hb) release as a function of shear stress and shearing time. Near the needle wall, velocities are low and shear stresses high, resulting in a significantly higher level of cell damage: 0.1% vs 0.001% in bulk flow for a mean flow of 91 ml/min into a 14G needle with a peak velocity of 220 cm/sec. The deviation from the classic Poiseuille velocity profile is shown. Less than 5% of the flow passes through this high damage path. A vortex at the inner side of the needle has a cumulative damage of 0.007% per 0.23 sec trip around the vortex.

Blood Flow Velocity↗