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Results for “Cell Tracking”

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PubMed1992

Circadian variations in cell cycle phase progression of mouse epidermal cells measured directly by bivariate BrdUrd/DNA flow cytometry.

Circadian stage-dependent variations in cell cycle traverse of mouse epidermal cells in vivo were investigated. The fate of cohorts of basal cells pulse-labelled with bromodeoxyuridine (BrdUrd) at different times of the day were studied by bivariate BrdUrd/DNA flow cytometry of isolated epidermal basal cells. Basal cells were tracked through the cell cycle up to 96 h after intraperitoneal injection of BrdUrd at 0800 and 2000, or followed for 6 h after BrdUrd injection at 0400, 1200, 1600 and 2400. The results confirmed our previous assumption that the cell cycle progression through S phase and G2 phase is considerably delayed at night, i.e. from 1600 to 0400, compared with daytime. The results indicate variations in G1 phase as well. The data strongly support the hypothesis that the main parameters responsible for circadian fluctuations in mitotic activity are variations in the S and G2 phase durations. The data are also consistent with the notion of proliferative heterogeneity among basal cells as described by a hierarchical proliferation model.

Animals↗

PubMed2007

[Effect of bone marrow stromal cells transfected with interleukin 18 on growth of intracranial glioma in rats].

BACKGROUND & OBJECTIVE: Because of the invasion and immune escape characteristics, surgical resection, radiotherapy, and chemotherapy had no definite curative effects on intracranial glioma. Because bone marrow stromal cells (BMSCs) can track migrating cells, and interleukin 18 (IL-18) can enhance antitumor immune reaction, this study was to explore the effect of IL-18-transfected BMSCs on growth of glioma in rats. METHODS: Pure BMSCs were obtained by culturing rat bone marrow cells and identified by flow cytometry (FCM). BMSCs were transfected with retrovirus LXSN/IL-18 to prepare BMSCs/IL-18. IL-18 genetic transcription and expression were assessed by reverse transcription-polymerase chain reaction (RT-PCR) and immunofluorescence. The characteristic variations of BMSCs were detected by MTT assay, FCM, and immunofluorescence. The effect of BMSCs/IL-18 on activation of T cells was evaluated by ELISA. Glioma-bearing rats were divided into BMSCs group, BMSCs/IL-18 group, PBS group, and control group and received relevant treatments. The effect of BMSCs/IL-18 on growth of glioma was observed. RESULTS: IL-18 gene was expressed stably in BMSCs/IL-18. The proliferation speed of BMSCs/IL-18 was slower than that of BMSCs. The secretion of interferon-gamma (IFN-gamma) from rat spleen lymphocytes in BMSCs/IL-18 group was 9 times more than that in BMSCs group. Tumor volume was (18.26+/-6.84) mm(3) in BMSCs group, (6.37+/-1.52) mm(3) in BMSCs/IL-18 group, (22.48+/-6.02) mm(3) in PBS group, and (21.06+/-5.83) mm(3) in control group; survival time of the rats was (25.3+/-6.4) days, (84.7+/-16.3) days, (21.6+/-4.7) days, and (22.5+/-6.2) days, respectively. After transplantation of BMSCs/IL-18, CD4(+) T cells in glioma were increased to 37.7+/-3.5 and CD8(+) T cells were increased to 32.3+/-4.5 in each field of view (x200). CONCLUSION: BMSCs/IL-18 could express IL-18 gene stably, and have definite therapeutic effect on glioma in rats.

Animals↗

PubMed1997

Enhanced green fluorescent protein as selectable marker of retroviral-mediated gene transfer in immature hematopoietic bone marrow cells.

The further improvement of gene transfer into hematopoietic stem cells and their direct progeny will be greatly facilitated by markers that allow rapid detection and efficient selection of successfully transduced cells. For this purpose, a retroviral vector was designed and tested encoding a recombinant version of the Aequorea victoria green fluorescent protein that is enhanced for high-level expression in mammalian cells (EGFP). Murine cell lines (NIH 3T3, Rat2) and bone marrow cells transduced with this retroviral vector demonstrated a stable green fluorescence signal readily detectable by flow cytometry. Functional analysis of the retrovirally transduced bone marrow cells showed EGFP expression in in vitro clonogenic progenitors (GM-CFU), day 13 colony-forming unit-spleen (CFU-S), and in peripheral blood cells and marrow repopulating cells of transplanted mice. In conjunction with fluorescence-activated cell sorting (FACS) techniques EGFP expression could be used as a marker to select for greater than 95% pure populations of transduced cells and to phenotypically define the transduced cells using antibodies directed against specific cell-surface antigens. Detrimental effects of EGFP expression were not observed: fluorescence intensity appeared to be stable and hematopoietic cell growth was not impaired. The data show the feasibility of using EGFP as a convenient and rapid reporter to monitor retroviral-mediated gene transfer and expression in hematopoietic cells, to select for the genetically modified cells, and to track these cells and their progeny both in vitro and in vivo.

Animals↗

PubMed2003

In vivo tracking of neural progenitor cell migration to glioblastomas.

The ability to noninvasively track the migration, engraftment, and proliferation of neural progenitor cells (NPCs) has significant clinical and research implications. The purpose of our study was to explore the macroscopic migratory capabilities of NPCs toward brain tumors after implantation into nude mice. We stably transfected C17.2 NPCs with the firefly luciferase gene (F-luc) and implanted cells into (1) the contralateral brain parenchyma (2 x 10(6) cells), (2) the ventricles (2 x 10(6) cells), (3) the vasculature (1 x 10(5) cells), or (4) the intraperitoneal cavity (5 x 10(6) cells) of mice bearing intracranial gliomas (Gli36). Using serial bioluminescence imaging, migration of parenchymally injected cells was observed across the corpus callosum, first detected at 1 week, with maximal density at the tumor site 2-3 weeks after implantation. Similar patterns were also observed with intraventricular injections; however, tumors were populated earlier, presumably because of the shorter distance to travel. Intravenous injections resulted in more modest tumoral NPC populations, whereas virtually no cells could be identified in tumors after intraperitoneal injection. These results confirm the migratory capability of NPCs over considerable distances and their preferential accumulation in brain tumors on CNS rather than peripheral injection.

Animals↗

PubMed1990

Track structure, lesion development, and cell survival.

A stochastic track-structure-dependent model is presented based on DNA double-strand breaks (DSBs) interacting in a time- and distance-dependent manner, and in competition with DSB repair, to form exchange-type chromosomal aberrations. Many models of cell survival involve estimation of mean numbers of lesions per cell, which is then related to cell survival. Unless this relationship is linear, this implies that a cell responds not to the number of lesions produced in it, but to the mean number of lesions in all the exposed cells; this is clearly unrealistic, particularly for phenomena such as saturation. In contrast to such deterministic approaches, we describe a stochastic model, in which individual cells are considered and exposed to Monte Carlo-generated tracks of various radiations. The elementary sublesions produced (DSBs) diffuse, repair, or interact, forming lesions (chromosomal exchange-type aberrations) in a time- and distance-dependent manner. Results agree well with experiments for survival of synchronous Chinese hamster V-79 cells exposed to X rays and radiations with LETs from 20 to 170 keV/microns. Thus the main features of survival for low-, medium-, and high-LET radiation are understandable in terms of a single approach, the relative responses to different radiations being determined by their different energy deposition patterns.

Cell Survival↗

PubMed2003

Clonal tracking of autoaggressive T cells in polymyositis by combining laser microdissection, single-cell PCR, and CDR3-spectratype analysis.

Clonal expansions of CD8+ T cells have been identified in muscle and blood of polymyositis patients by PCR techniques, including T cell receptor (TCR) complementarity-determining region (CDR)3 length analysis (spectratyping). To examine a possible pathogenic role of these clonally expanded T cells, we combined CDR3 spectratyping with laser microdissection and single-cell PCR of individual myocytotoxic T cells that contact, invade, and destroy a skeletal muscle fiber. First, we screened cDNA from muscle biopsy specimens by CDR3 spectratyping for expanded TCR beta chain variable region (BV) sequences. To pinpoint the corresponding T cells in tissue, we stained cryostat sections with appropriate anti-TCR BV mAbs, isolated single BV+ T cells that directly contacted or invaded a muscle fiber by laser-assisted microdissection, and amplified their TCR BV chain sequences from rearranged genomic DNA. In this way, we could relate the oligoclonal peaks identified by CDR3-spectratype screening to morphologically characterized microdissected T cells. In one patient, a large fraction of the microdissected T cells carried a common TCR-BV amino acid CDR3 motif and conservative nucleotide exchanges in the CDR3 region, suggesting an antigen-driven response. In several cases, we tracked these T cell clones for several years in CD8+ (but not CD4+) blood lymphocytes and in two patients also in consecutive muscle biopsy specimens. During immunosuppressive therapy, oligoclonal CDR3-spectratype patterns tended to revert to more polyclonal Gaussian distribution-like patterns. Our findings demonstrate that CDR3 spectratyping and single-cell analysis can be combined to identify and track autoaggressive T cell clones in blood and target tissue. This approach should be applicable to other inflammatory and autoimmune disorders.

Adult↗

PubMed2005

Position, direction of movement, and speed tuning of cerebellar Purkinje cells during circular manual tracking in monkey.

The cerebellum plays an essential role in pursuit tracking with the eye and with the hand. During smooth pursuit eye movements, both tracking position and velocity are signaled by Purkinje cells. Purkinje cell simple spike discharge is also modulated by direction and speed during linear manual tracking. This study evaluated how all three parameters, position, movement direction, and speed, are signaled in the simple spike discharge of Purkinje cells during circular manual tracking. Three rhesus monkeys intercepted and then tracked a target moving in a circle in both counterclockwise and clockwise directions across a range of constant target speeds. Two sets of analyses of the simple spike firing of 97 Purkinje cells examined the effects of position, movement direction, and speed. The first approach was the incremental improvement of regression models, initially modeling a pure position dependence, then incorporating movement direction, and finally incorporating speed dependence. The second was a model-independent approach, without any explicit assumptions about the character of the directional tuning or speed effects. Both analyses revealed the same three results: (1) Purkinje cell discharge is spatially tuned, to both the position and direction of movement, and (2) this spatial tuning is not altered by the speed, except (3) the speed scales the average firing and/or depth of modulation. The results suggest that the population of Purkinje cells forms a representation of the entire position-direction space of arm movements, and that the speed modulates the scale of that representation. This speed scaling provides insights into the cerebellar processing of movement-related timing.

Action Potentials↗

PubMed2005

Analysis of green fluorescent protein expression in transgenic rats for tracking transplanted neural stem/progenitor cells.

Green fluorescent protein (GFP) expression was evaluated in tissues of different transgenic rodents--Sprague-Dawley (SD) rat strain [SD-Tg(GFP)Bal], W rat strain [Wistar-TgN(CAG-GFP)184ys], and M mouse strain [Tg(GFPU)5Nagy/J]--by direct fluorescence of native GFP expression and by immunohistochemistry. The constitutively expressing GFP transgenic strains showed tissue-specific differences in GFP expression, and GFP immunohistochemistry amplified the fluorescent signal. The fluorescence of stem/progenitor cells cultured as neurospheres from the ependymal region of the adult spinal cord from the GFP SD and W rat strains was assessed in vitro. After transplantation of the cells into wild-type spinal cord, the ability to track the grafted cells was evaluated in vivo. Cultured stem/progenitor cells from the SD strain required GFP immunostaining to be visualized. Likewise, after transplantation of SD cells into the spinal cord, immunohistochemical amplification of the GFP signal was required for detection. In contrast, GFP expression of stem/progenitor cells generated from the W strain was readily detected by direct fluorescence both in vitro and in vivo without the need for immunohistochemical amplification. The cultured stem/progenitor cells transplanted into the spinal cord survived for at least 49 days after transplantation, and continued to express GFP, demonstrating stable expression of the GFP transgene in vivo.

Animals↗

PubMed2005

Automated method for tracking individual red blood cells within capillaries to compute velocity and oxygen saturation.

OBJECTIVE: The authors present a new method to track individual red blood cells (RBCs) as they move through capillaries. This method uses a recently developed Measurement and Analysis System for Capillary Oxygen Transport (MASCOT) and the concept of space-time images to track RBCs between consecutive frames of video recordings of the microcirculation. METHODS: A space-time image displays in a single static image for a single capillary the location of all RBCs as a function of time. Analysis is performed on video tapes of RBC flow through capillaries to obtain velocity of individual cells as they traverse the capillary of interest. A space-time image is generated to track RBCs from one frame to the next and their velocities are computed. Based on the optical density values of each cell obtained from synchronized videotapes at two wavelengths, the oxygen saturation of a cell can be determined. In this manner, oxygen saturation can be tracked for the same cells as they move through the capillary. RESULTS AND CONCLUSIONS: These measurements, taken together, allow one to determine how much and how fast oxygen is being delivered to the surrounding tissue. This method provides, for the first time, a way to track individual RBCs flowing through capillary networks and study their RBC dynamics and oxygenation.

Algorithms↗

PubMed2004

In vivo tracking of implanted stem cells using radio-labeled transferrin scintigraphy.

The possibility of monitoring stem cells in vivo with radionuclide imaging after transplantation was investigated. Based on the results of a radioligand receptors assay that human mesenchymal stem cells (hMSCs) express a high level of transferrin receptors, iodinated transferrin (131I-Tf(Fe)2) was chosen as the radiotracer for imaging the cells implanted into the spinal cords of rabbits. Accumulation of radioactivity at the cell transplanted sites was assessed 16 and 24 hours post-intrathecal injection of 131I-Tf(Fe)2. Transferrin receptors expression and Tf binding of the implanted cells were verified by immunofluorescence and ex vivo phosphor imaging. The specificity of Tf uptake of hMSCs was proved through control experiments, i.e., replacing 131I-Tf(Fe)2 with 131I labeled human serum albumin as the tracer or substituting hMSCs with phosphate buffered saline as the grafts. Despite some defects, such as the invasive administration of the tracer and the non-specificity of transferrin receptors as a marker of stem cells in this preliminary study, the technique of nuclear medicine imaging is considered to have great potential in tracking implanted cells in vivo.

Animals↗

PubMed2003

Lentivector-mediated clonal tracking reveals intrinsic heterogeneity in the human hematopoietic stem cell compartment and culture-induced stem cell impairment.

Knowledge of the composition and interrelationship of the various hematopoietic stem cells (HSCs) that comprise the human HSC pool and the consequence of culture on each class is required for effective therapies based on stem cells. Clonal tracking of retrovirally transduced HSCs in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice revealed heterogeneity in the repopulation capacity of SCID-repopulating cells (SRCs). However, it is impossible to establish whether HSC heterogeneity is intrinsic or whether the culture conditions required for retroviral transduction induce qualitative and quantitative alterations to SRCs. Here, we report establishment of a clonal tracking method that uses lentivectors to transduce HSCs with minimal manipulation during overnight culture without cytokine stimulation. By serial bone marrow (BM) sampling of mice receiving transplants, short-term SRCs (ST-SRCs) and long-term SRCs (LT-SRCs) were identified on the basis of repopulation dynamics demonstrating that their existence is not an experimental artifact but reflects the state of the HSC pool. However, 4 days of culture in conditions previously used for SRC retroviral transduction significantly reduced SRC number as assessed by clonal analysis. These studies provide a foundation to understand the molecular and cellular determinants of human HSC development and to develop therapies targeted to specific HSC classes.

Animals↗

PubMed1985

Do cells show an inverse locomotory response to fibronectin and laminin substrates?

Sixteen cell types from a variety of tissues and from primary and secondary cell cultures and established cell lines were tested for their ability to distinguish between fibronectin and laminin substrates during locomotion in vitro. Laminin and fibronectin were presented to the cells as directly adjacent tracks. Most cells, regardless of origin, showed no preference for one substrate over the other. Only two of the cell types tested showed a strong preference for one or other other substrate molecule. Cells were responding to the local substrate, since antibodies directed against one substrate molecule only interfered with locomotion on tracks coated with that molecule. We conclude that many cells simultaneously express functionally active receptors for fibronectin and laminin, and that differential locomotory response to these two molecules cannot be assumed without experimental confirmation.

Animals↗

PubMed2002

Visualization of the intracellular behavior of HIV in living cells.

To track the behavior of human immunodeficiency virus (HIV)-1 in the cytoplasm of infected cells, we have tagged virions by incorporation of HIV Vpr fused to the GFP. Observation of the GFP-labeled particles in living cells revealed that they moved in curvilinear paths in the cytoplasm and accumulated in the perinuclear region, often near the microtubule-organizing center. Further studies show that HIV uses cytoplasmic dynein and the microtubule network to migrate toward the nucleus. By combining GFP fused to the NH2 terminus of HIV-1 Vpr tagging with other labeling techniques, it was possible to determine the state of progression of individual particles through the viral life cycle. Correlation of immunofluorescent and electron micrographs allowed high resolution imaging of microtubule-associated structures that are proposed to be reverse transcription complexes. Based on these observations, we propose that HIV uses dynein and the microtubule network to facilitate the delivery of the viral genome to the nucleus of the cell during early postentry steps of the HIV life cycle.

Biological Transport↗

PubMed2005

Tracking hematopoiesis at the single cell level.

Despite intensive research, many longstanding questions of experimental hematology remain unsolved. One major reason is the fact that hematopoiesis is usually followed by analyzing populations of cells rather than individual cells, at few points in time during an experiment and without knowing (or quickly loosing) the cells' individual identities. The static picture yielded by this approach makes it impossible to appreciate the dynamic developmental processes leading to the generation of the full hematopoietic system from individual hematopoietic stem cells (HSCs). Real-time tracking of individual cells in culture, tissues, or whole organisms would be an extremely powerful approach to fully understand the developmental complexity of hematopoiesis. To this end, a computer-aided culture and bioimaging system is being developed to follow the fate of individual cells over long periods of time. This system is used to follow the development of multilineage cobblestone colonies from adult HSCs in stroma cocultures at the single cell level over many generations. To facilitate noninvasive detection of lineage commitment in these cultures, new subcellular forms of optimized fluorescent proteins have been developed to allow simultaneous marking of multiple hematopoietic lineages within the same animal.

Adult↗

PubMed2004

In vivo magnetic resonance imaging tracks adult neural progenitor cell targeting of brain tumor.

Using magnetic resonance imaging (MRI), we described a method for noninvasively tracking grafted neural progenitor cells and bone marrow stromal cells (MSCs) in brain tumor of the rat. Neural progenitor cells and MSCs were labeled with lipophilic dye-coated superparamagnetic particles. The labeled neural progenitor cells and MSCs were transplanted to rats via the cisterna magna and a tail vein, respectively, 1 week after 9L-gliosarcoma cell implantation. Three-dimensional (3D) gradient echo and contrast agent images revealed dynamic migration of adult neural progenitor cells and MSCs detected by loss of MRI signals towards tumor mass and infiltrated tumor cells. Prussian blue staining and fluorescent microscope analysis showed that grafted cells targeted tumor cells and areas with grafted cells corresponded to areas with loss of MRI signals. These results demonstrate that the MRI technique provides a sensitive method for in vivo assessment of grafted cells targeting tumor mass and infiltrated tumor cells and that adult neural progenitor cells and MSCs can target tumor aggregates in the brain.

Animals↗

PubMed1998

Acid-induced responses in hamster chorda tympani and intracellular pH tracking by taste receptor cells.

HCl- and NaCl-induced hamster chorda tympani nerve responses were recorded during voltage clamp of the lingual receptive field. Voltage perturbations did not influence responses to HCl. In contrast, responses to NaCl were decreased by submucosal-positive and increased by submucosal-negative voltage clamp. Responses to HCl were insensitive to the Na+ channel blockers, amiloride and benzamil, and to methylisobutylamiloride (MIA), an Na+/H+ exchange blocker. Responses to NaCl were unaffected by MIA but were suppressed by benzamil. Microfluorometric and imaging techniques were used to monitor the relationship between external pH (pHo) and the intracellular pH (pHi) of fungiform papilla taste receptor cells (TRCs) following 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein loading. TRC pHi responded rapidly and monotonically to changes in pHo. This response was unaffected by Na+ removal or the presence of amiloride, benzamil, or MIA. The neural records and the data from isolated TRCs suggest that the principal transduction pathway for acid taste in hamster is similar to that in rat. This may involve the monitoring of changes in TRC pHi mediated through amiloride-insensitive H+ transport across TRC membranes. This is an example of cell monitoring of environmental pH through pH tracking, i.e., a linear change in pHi in response to a change in pHo, as has been proposed for carotid bodies. In taste, the H+ transport sites may be concentrated on the basolateral membranes of TRCs and, therefore, are responsive to an attenuated H+ concentration from diffusion of acids across the tight junctions.

Amiloride↗

PubMed2004

Diet and chronic haloperidol effects on rat midbrain dopamine neurons.

The effects of dietary glucose (chow containing 0%, 10%, 20%, or 40% glucose, w/w) on chronic haloperidol-induced changes in dopamine (DA) neuronal activity were tested. Rats were treated daily by oral gavage for 21 days with either water or 0.5 mg/kg haloperidol, then anesthetized for in vivo electrophysiological recording. The numbers of spontaneously active DA neurons in the substantia nigra (A9) and ventral tegmental area (A10) regions of the midbrain were estimated with the cells-per-track sampling method. In rats fed standard chow, haloperidol significantly reduced the number of active neurons in both regions compared to water controls. In water controls there were no differences in DA cells per track between rats fed standard chow or chow containing 10% or 20% glucose, whereas these glucose diets significantly attenuated the effects of chronic haloperidol on DA cells per track. The 40% glucose diet itself nonsignificantly reduced cells per track and, in turn, nonsignificantly attenuated the effects of haloperidol. The results demonstrate that dietary glucose content can alter haloperidol-induced changes in the activity of midbrain DA neurons.

Action Potentials↗

PubMed2003

Organotypic slices in vitro: repeated, same-cell, high-resolution tracking of nuclear and cytoplasmic fluorescent signals in live, transfected cerebellar neurons by confocal microscopy.

The culture of organotypic slices for the purposes of tracking dynamic cellular events within the same live cell at high resolution, as a function of development in vitro has not been previously reported. The present study was undertaken to define the conditions most suitable for both the in vitro organotypic development of Purkinje neurons in cerebellar slices of neonatal mice, and the repeated visualization of nuclear signals within such cells. Slices of cerebella were maintained on 25 mm diameter, collagen-coated Anodisc membranes, placed in six-well plates and raised to the air-medium interface by use of glass fibre filter supports. This system permits cultures to be repeatedly observed both by phase contrast microscopy and, upon biolistic transfection, by laser confocal microscopy using 40x, 60x, and 100x water-immersion objectives, at high resolution. Upon co-transfection with two plasmids, differentiation of the same transfected Purkinje neurons was followed across in vitro development for periods of up to 10 days. Despite the relative thickness of the slice culture, even small, punctate, nuclear signals, were detectable. The results show that Purkinje neurons in cerebellar slices explanted from postnatal day 2 mice, developed cytotypically, although some were ectopically located. In contrast, Purkinje neurons in slices from postnatal day 6 cerebella developed in an organotypic manner. It is concluded that this culture system serves as an ideal tool for applications in experimental biology where high resolution tracking of cellular signals, over extended time periods, is of interest.

Animals↗