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Results for “Cell Tracking”

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PubMed2001

Effects of antibody concentration on the separation of human natural killer cells in a commercial immunomagnetic separation system.

BACKGROUND: The magnetic separation of a cell population based on cell surface markers is a critical step in many biological and clinical laboratories. In this study, the effect of antibody concentration on the separation of human natural killer cells in a commercial, immunomagnetic cell separation system was investigated. METHODS: Specifically, the degree of saturation of antibody binding sites using a two-step antibody sandwich was quantified. The quantification of the first step, a primary anti-CD56-PE antibody, was achieved through fluorescence intensity measurements using a flow cytometer. The quantification of the second step, an anti-PE-microbeads antibody reagent, was achieved through magnetophoretic mobility measurements using cell tracking velocimetry. RESULTS: From the results of these studies, two different labeling protocols were used to separate CD56+ cells from human, peripheral blood by a Miltenyi Biotech MiniMACS cell separation system. The first of these two labeling protocols was based on company recommendations, whereas the second was based on the results of the saturation studies. The results from these studies demonstrate that the magnetophoretic mobility is a function of both primary and secondary antibody concentrations and that mobility does have an effect on the performance of the separation system. CONCLUSIONS: As the mobility increased due to an increase in bound antibodies, the positive cells were almost completely eliminated from the negative eluent. However, with an increase in bound antibodies, and thus mobility, the total amount of positive cells recovered decreases. It is speculated that these cells are irreversibly retained in the column. These results demonstrate the complexity of immunomagnetic cell separation and the need to further optimize the cell separation process.

Antigen-Antibody Reactions↗

PubMed1989

Kinetics of changes in peritoneal cell populations following acute inflammation.

The kinetics of macrophage (M phi) recruitment to the peritoneum following the induction of acute inflammation by thioglycollate broth (TG) was evaluated after prelabeling resident M phi with the fluorescent cell tracking dye, PKH-1. Most of the PKH-1-labeled resident M phi disappeared from the recoverable peritoneal cell population within the first hour after injection of TG. This disappearance coincided with the inflammatory influx of neutrophils (PMNs) and was sustained for at least 5 days after administration of TG, although the PMN number had returned to resident levels by this time. PKH-1-labeled peritoneal M phi were observed again in most animals at 7 days after injection of TG. The number of labeled M phi recovered at 7 days was approximately twice the number of resident peritoneal M phi in control animals which did not receive the TG broth. These additional M phi may include progeny of either the resident M phi or other local M phi precursors, such as omental M phi, which were labeled by the PKH-1 injection.

Animals↗

PubMed1994

Spectra of fluorescent dyes used in flow cytometry.

Flow cytometry uses a relatively small set of dyes for immunochemistry and nucleic acid detection, most of which have been known and used reliably in flow cytometry for several years. These can usually be combined to make simultaneous two-color measurements of multiple cell-surface antigens and nucleic acid content. Because of the overlap of dye spectra and difficulties in finding dyes with substantial Stokes shifts that can be excited by the argon laser, simultaneous three-color or more detection can be more difficult. A basic knowledge of the factors that go into producing the fluorescent signal, including the spectra of dyes and their overlap, is necessary in planning multicolor experiments. By contrast, there have been a number of new fluorescent probes developed for detecting ions, membrane potential, metabolism, organelles, and other properties of living cells as well as for determining cell viability, proliferation, and cell tracking. So far most of these physiological probes are being used only for fundamental research rather than for cell classification. However, as research activity expands in this area, its diagnostic potential is likely to be increasingly appreciated.

Animals↗

PubMed2005

Cell sorting by one gravity SPLITT fractionation.

The need for innovative separative techniques suitable for the fractionation of biomaterials prompted this investigation into the performance of the gravitational split-flow thin channel (G-SPLITT) system as a cell sorter. The rigorous mathematical description of the separation mechanism allows achievement of fast separation of several million myeloma cells from healthy splenocytes using flow conditions calculated from theory. Separation in G-SPLITT is based on differences in sedimentation rate. For accurate prediction of the optimal working conditions, this parameter was directly measured by cell tracking velocimetry rather than relying on a measure of diameter (by Multisizer) and an assumed density for each cell population. We also discuss the influence of different flow conditions on the effectiveness of separation.

Cell Separation↗

PubMed2004

'Proliferative' and 'synthetic' airway smooth muscle cells are overlapping populations.

The extension of airway smooth muscle cell (ASMC) functions, from just contractile, to synthetic and/or proliferative states, is an important component of airway remodelling and inflammation in asthma. Whereas all these functions have been demonstrated in ASM, currently, it is not known whether ASMC can be differentiated on the basis of their proliferative and synthetic functions. We used flow-cytometric techniques to determine, first, whether human ASMC are phenotypically heterogenous with regard to their secretory function, and second, the proliferative status of secretory cells. ASMC were induced to synthesize GM-CSF by stimulation with IL-1beta and TNF-alpha followed by 10% human serum. Flow-cytometric detection of intracellular GM-CSF revealed that only a proportion of cells in culture (approximately 20-60%) synthesize GM-CSF. To determine the proliferative status of GM-CSF producing cells, ASMC were pretreated with 5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE), a fluorescein based dye used to track cell division, prior to cytokine/serum stimulation. Simultaneous analysis of intracellular GM-CSF and CFSE revealed that GM-CSF producing cells were present in both the divided and undivided ASMC populations. Thus, cytokine production and proliferation occurred in overlapping ASMC populations and prior progression through the cell cycle was not essential for ASMC cytokine production.

Bronchi↗

PubMed2025

Measuring Cell Dimensions in Fission Yeast Using Machine Learning.

In fission yeast (Schizosaccharomyces pombe), cell length is a crucial indicator of cell cycle progression. Microscopy screens that examine the effect of agents or genotypes suspected of altering genomic or metabolic stability and thus cell size are crucial for studying disruptions to cell cycle dynamics. This method is based on using an automated cell segmentation algorithm to measure S. pombe cells imaged by brightfield (BF) microscopy methods. PhotoPhenosizer (PP) is a machine learning-based tool designed for automated cell measuring and dimensional analysis of morphology frequency distributions. Integration of this method into large-scale pipelines for tracking cell dimension change streamlines morphological measurements, which facilitates the examination of cellular responses to genomic and metabolic stresses. In this protocol, we use PP to observe the effect of genomic instability on cell size dynamics over a 12-day chronological lifespan assay. Our results show that relative to wild-type cells, a replication stress mutant shows larger cells during chronological aging in excess glucose media. Our results are consistent with activation of checkpoints that regulate cell morphology in response to DNA damage. This method's application highlights the relevance of its incorporation in experimental routines that require large-scale image processing and its adoption by users with routine needs in S. pombe molecular research projects.

Schizosaccharomyces↗

PubMed1994

Dispersive locomotion of human neutrophils in response to a steroid-induced factor from monocytes.

A monocyte-derived factor that stimulates the locomotion of human neutrophils on an albumin-coated glass surface has been prepared from the culture supernatant of dexamethasone-treated human monocytes and called STMS (steroid-treated monocyte supernatant). A modified cell tracking program has been developed and the parameters of locomotion determined by the analysis of Gail and Boone for cells moving in a persistent random walk. Cells moving in uniform concentrations of STMS, interleukin-8 (IL-8) and N-formyl-methionyl-leucyl-phenylalanine (fMLP) chosen to give a sub-maximal speed of locomotion show persistent, random and constrained random diffusion, respectively, with augmented diffusion coefficients of 0.8 +/- 0.1, 0.14 +/- 0.02 and 0.12 +/- 0.03 microns 2 per second for STMS, IL-8 and fMLP, respectively. The augmented diffusion coefficient and the underlying persistence are therefore sensitive quantitative assay parameters for STMS activity and the qualitative characteristics of locomotion allow STMS activity to be distinguished from that of all other factors tested. The contribution of lowered adhesion to locomotion was examined in a novel tilt-assay, which demonstrated that cells in the presence of STMS, but not other factors, moved down slope with significantly increased speed while maintaining contact with the substratum. The results were interpreted in terms of the bipolar form of STMS-treated cells, contrasting with multipolar forms in response to other agents. This together with low adhesiveness plus an inherent tendency of a single locomotor focus to continue motion in its original direction has been used to explain the difference between response to STMS and other factors.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell Adhesion↗

PubMed2006

Binding affinities/avidities of antibody-antigen interactions: quantification and scale-up implications.

Bioaffinity interactions have been, and continue to be, successfully adapted from nature for use in separation and detection applications. It has been previously reported that the magnetophoretic mobility of labeled cells show a saturation type phenomenon as a function of the concentration of the free antibody-magnetic nanoparticle conjugate which is consistent with other reports of antibody-fluorophore binding. Starting with the standard antibody-antigen relationship, a model was developed which takes into consideration multi-valence interactions, and various attributes of flow cytometry (FCM) and cell tracking velocimetry (CTV) measurements to determine both the apparent dissociation constant and the antibody-binding capacity (ABC) of a cell. This model was then evaluated on peripheral blood lymphocytes (PBLs) labeled with anti CD3 antibodies conjugated to FITC, PE, or DM (magnetic nanoparticles). Reasonable agreements between the model and the experiments were obtained. In addition, estimates of the limitation of the number of magnetic nanoparticles that can bind to a cell as a result of steric hinderance was consistent with measured values of magnetophoretic mobility. Finally, a scale-up model was proposed and tested which predicts the amount of antibody conjugates needed to achieve a given level of saturation as the total number of cells reaches 10(10), the number of cells needed for certain clinical applications, such as T-cell depletions for mismatched bone marrow transplants.

Antibody Affinity↗

PubMedDate not supplied

Separation of a breast cancer cell line from human blood using a quadrupole magnetic flow sorter.

We have developed a quadrupole magnetic flow sorter (QMS) to facilitate high-throughput binary cell separation. Optimized QMS operation requires the adjustment of three flow parameters based on the immunomagnetic characteristics of the target cell sample. To overcome the inefficiency of semiempirical operation/optimization of QMS flow parameters, a theoretical model of the QMS sorting process was developed. Application of this model requires measurement of the magnetophoretic mobility distribution of the cell sample by the cell tracking velocimetry (CTV) technique developed in our laboratory. In this work, the theoretical model was experimentally tested using breast carcinoma cells (HCC1954) overexpressing the HER-2/neu gene, and peripheral blood leukocytes (PBLs). The magnetophoretic mobility distribution of immunomagnetically labeled HCC1954 cells was measured using the CTV technique, and then theoretical predictions of sorting recoveries were calculated. Mean magnetophoretic mobilities of (1-3) x 10(-4) mm(3)/(T A s) were obtained depending on the labeling conditions. Labeled HCC1954 cells were mixed with unlabeled PBLs to form a "spiked" sample to be separated by the QMS. Fractional recoveries of cells for different flow parameters were examined and compared with theoretical predictions. Experimental results showed that the theoretical model accurately predicted fractional recoveries of HCC1954 cells. High-throughput (3.29 x 10(5) cells/s) separations with high recovery (0.89) of HCC1954 cells were achieved.

Algorithms↗

PubMed2003

Labeling of hematopoietic stem and progenitor cells in novel activatable EGFP reporter mice.

Conditional activation and inactivation of genes using the Cre/loxP recombination system is a powerful tool for the analysis of gene function and for tracking cell fate. Here we report a novel silent EGFP reporter mouse line generated by enhancer trap technology using embryonic stem (ES) cells. Following transfection with the silent EGFP reporter construct, positive ES cell clones were treated with Cre recombinase. These "activated clones" were then further selected on the basis of ubiquitous EGFP expression during in vitro differentiation. The parental "silent" clones were then used for generating mice. Upon Cre-mediated activation in ovo tissues tested from these mice express EGFP. Long-term, strong and sustainable expression of EGFP is observed in most myeloid and lymphoid cells. As shown by in vivo transplantation assays, the majority of hematopoietic stem cells (HSCs) and spleen colony-forming units (CFU-S) reside within the EGFP positive fraction. Most in vitro colony-forming units (CFU-Cs) isolated from bone marrow also express EGFP. Thus, these reporter mice are useful for the analysis of Cre-mediated recombination in HSCs and hematopoietic progenitor cells. This, in combination with the high accessibility of the loxP sites, makes these mice a valuable tool for testing cell/tissue-specific Cre-expressing mice. .

Animals↗

PubMedDate not supplied

Computer tracking of germ cells in the cycle of the seminiferous epithelium and prediction of changes in cycle duration in animals commonly used in reproductive biology and toxicology.

A computer program called Stages was written to aid the tracking of germ cells and stages forward and backward through time in the cycle of the seminiferous epithelium. The program incorporates the basic kinetics of spermatogenesis in the rat, mouse, hamster, guinea pig, dog, rabbit, bull, stallion, ram, boar, quail, monkey, and man. The program is flexible and permits the user to alter the cycle duration time and the frequency of each stage. Compiled for use on personal computers and available on floppy disks free of charge, Stages is menu-driven and requires no knowledge of programming. The program was tested using published data of testicular toxicity and vitamin A synchronization of stages. In general, predicted cell types were similar to those observed; discrepancies between observed and predicted data are discussed. When cycle duration time was changed, predicted data for stage synchronization coincided with the observed data. This program will improve the speed and accuracy of studying factors that affect spermatogenesis. By using Stages, it is possible to predict the target cell types in a toxicity study and to track affected cells over long periods of time. Predicted cell types may also indicate which cells to examine biochemically as well as morphologically in subsequent experiments. The program will also be useful to beginning students learning the complex patterns of cellular associations and the progression of germ cells in the cycle of the seminiferous epithelium.

Animals↗

PubMed2000

High-efficiency retroviral transduction of mammalian cells on positively charged surfaces.

The efficiency of retroviruses as transducing agents has been appreciated for many years, particularly for hematopoietic cell targets for which alternative strategies applicable to adherent cells are not effective. Advances in vector design, pseudotyping, and infection conditions have eliminated the need to cocultivate the target cells with virus-producing cells. Nevertheless, improvements are still needed for many applications, including those with a therapeutic or clinical cell-tracking objective. In this study we show that more positively charged surfaces, including those designed for the culture of anchorage-dependent cells, allow measurable levels of adhesion by different pseudotypes of retroviruses, which can result in increased gene transfer efficiencies to a variety of target cells including normal primary human hematopoietic cells as well as human leukemic cell lines and rat and murine fibroblasts. In the experiments with primary human cells, equal aliquots of enriched CD34+ cord blood cells were first stimulated for 2 days with cytokines (Flt3 ligand, Steel factor, IL-3, IL-6, and G-CSF) and then exposed for 4 days to a green fluorescent protein (GFP)- and Neo(r)-encoding retrovirus produced in PG13 cells. Both the final yield (approximately 300% relative to initial numbers), and the proportion (approximately 60%) of transduced CD34+ cells, colony-forming cells, and long-term culture-initiating cells were the same for cells infected either in tissue culture dishes or in fibronectin-coated petri dishes. Similar proportions (approximately 10%) and absolute yields of GFP+ human cells were also found in multilineage engrafted NOD/SCID mice assessed 6 to 8 weeks after being transplanted with these two types of transduced, but unselected, cells. These findings suggest a new and simpler approach for achieving high gene transfer efficiencies to hematopoietic cells.

Animals↗

PubMed1993

Involvement of rho p21 and its inhibitory GDP/GTP exchange protein (rho GDI) in cell motility.

Evidence is accumulating that rho p21, a ras p21-related small GTP-binding protein (G protein), regulates the actomyosin system. The actomyosin system is known to be essential for cell motility. In the present study, we examined the action of rho p21, its inhibitory GDP/GTP exchange protein (named rho GDI), its stimulatory GDP/GTP exchange protein (named smg GDS), and Clostridium botulinum ADP-ribosyltransferase C3, known to selectively ADP-ribosylate rho p21 and to impair its function, in cell motility (chemokinesis) of Swiss 3T3 cells. We quantitated the capacity of cell motility by measuring cell tracks by phagokinesis. Microinjection of the GTP gamma S-bound active form of rhoA p21 or smg GDS into Swiss 3T3 cells did not affect cell motility, but microinjection of rho GDI into the cells did inhibit cell motility. This rho GDI action was prevented by comicroinjection of rho GDI with the GTP gamma S-bound form of rhoA p21 but not with the same form of rhoA p21 lacking the C-terminal three amino acids which was not posttranslationally modified with lipids. The rho GDI action was not prevented by Ki-rasVal-12 p21 or any of the GTP gamma S-bound form of other small GTP-binding proteins including rac1 p21, G25K, and smg p21B. Among these small G proteins, rhoA p21, rac1 p21, and G25K are known to be substrates for rho GDI. The rho GDI action was not prevented by comicroinjection of rho GDI with smg GDS. Microinjection of C3 into Swiss 3T3 cells also inhibited cell motility. These results indicate that the rho GDI-rho p21 system regulates cell motility, presumably through the actomyosin system.

3T3 Cells↗

PubMed1993

The effect of the motility stimulants, caffeine, pentoxifylline, and 2-deoxyadenosine on hyperactivation of cryopreserved human sperm.

OBJECTIVE: To determine if the motility stimulants, caffeine (7 mM), pentoxifylline (3 mM), 2-deoxyadenosine (3 mM), and a combination of the three would induce hyperactivated (HA) motility. DESIGN: Controlled laboratory study of stimulants using cryopreserved semen from 10 donors at three time points. SETTING: The study was performed in the Andrology Laboratory at Prince Henry's Institute of Medical Research Clayton, Victoria, Australia. INTERVENTIONS: Stimulants in doses maximally effective for stimulation of motility were incubated with suspensions of previously cryopreserved sperm. MAIN OUTCOME MEASURES: Motility characteristics (curvilinear velocity [VCL], linearity [LIN], and maximum amplitude of lateral head displacement [ALHmax]) were derived using the single cell track facility of the CellSoft computer-automated semen analyzer (Cryo Resources Ltd., Montgomery, NY). Videotapes were visually inspected, and 125 sperm cell trajectories exhibiting characteristic HA behavior were identified. The HA motility thresholds (5th or 95th centiles) were as follows: VCL > 74 microns/s, LIN < 74%, and ALHmax > 4.7 microns. Cells with motility characteristics outside these limits were regarded as HA. The significance of the effect of the stimulants on the proportion of sperm exhibiting HA was examined by Poisson regression analysis. RESULTS: Sperm washing (removal of the cells from the cryoprotectant) and swim-up caused significant changes in the VCL and straight line velocity and a twofold (5.6% to 11.9%) increase in the proportion of HA cells. In the presence of motility stimulants, the proportion of HA cells was significantly increased threefold (11.9% to 32.5%) above that seen in the control washed sperm. Hyperactivated motility declined after 1 to 2 hours, but 2-deoxyadenosine demonstrated a prolonged effect. CONCLUSION: These motility stimulants that affect adenosine 3':5' monophosphate in human sperm stimulate cyclic hyperactivation.

Caffeine↗

PubMed2005

Androgenic switch: an example of plant embryogenesis from the male gametophyte perspective.

Embryogenesis in plants is a unique process in the sense that it can be initiated from a wide range of cells other than the zygote. Upon stress, microspores or young pollen grains can be switched from their normal pollen development towards an embryogenic pathway, a process called androgenesis. Androgenesis represents an important tool for research in plant genetics and breeding, since androgenic embryos can germinate into completely homozygous, double haploid plants. From a developmental point of view, androgenesis is a rewarding system for understanding the process of embryo formation from single, haploid microspores. Androgenic development can be divided into three main characteristic phases: acquisition of embryogenic potential, initiation of cell divisions, and pattern formation. The aim of this review is to provide an overview of the main cellular and molecular events that characterize these three commitment phases. Molecular approaches such as differential screening and cDNA array have been successfully employed in the characterization of the spatiotemporal changes in gene expression during androgenesis. These results suggest that the activation of key regulators of embryogenesis, such as the BABY BOOM transcription factor, is preceded by the stress-induced reprogramming of cellular metabolism. Reprogramming of cellular metabolism includes the repression of gene expression related to starch biosynthesis and the induction of proteolytic genes (e.g. components of the 26S proteasome, metalloprotease, cysteine, and aspartic proteases) and stress-related proteins (e.g. GST, HSP, BI-1, ADH). The combination of cell tracking systems with biochemical markers has allowed the key switches in the developmental pathway of microspores to be determined, as well as programmed cell death to be identified as a feature of successful androgenic embryo development. The mechanisms of androgenesis induction and embryo formation are discussed, in relation to other biological systems, in special zygotic and somatic embryogenesis.

Apoptosis↗

PubMed2004

Migration of antigen presenting cells from periphery to the peritoneum during an inflammatory response: role of chemokines and cytokines.

We demonstrate the migration of antigen presenting cells (APCs), macrophages, and dendritic cells from the subcutaneous site to the peritoneum after they have picked up the antigen, using cell tracking dye. The migration of the APCs is more universal as it was also observed after injection of MethA tumor, DH-5alpha cells, and leishmania parasites, in addition to AK-5 tumor cells. Cellular migration is mediated by several chemokines and cytokines that also induce heavy influx of immune cells into the peritoneum. MIP-3beta secreted by the mesothelial cells is involved in the cellular influx into the peritoneum, whereas IL-12 and IFN-gamma produced by the APCs induced activation of immune cells in the peritoneum. Our results suggest an antigen presentation function for the APCs in the peritoneum as studied by lymphoproliferation assays. These studies indicate antigen presentation function of the activated migratory APCs from the distant subcutaneous site to the peritoneum, suggesting it acts as an important lymphoid organ involved in the enhancement of effector cell function.

Antigens, Neoplasm↗

PubMed1997

Contact of sarcoma cells with aligned fibroblasts accelerates their displacement: computer-assisted analysis of tumour cell locomotion in co-culture.

The shape and locomotion of rat sarcoma XC cells on glass, polystyrene, and confluent monolayer cultures of aligned human skin fibroblasts were studied with quantitative, computer-assisted methods. The cell shape depended upon the substratum; the sarcoma cells seeded on fibroblasts assumed polarized shapes. The tumour cells emigrating from aggregates and in sparse cultures showed random locomotion when plated on glass or on the polystyrene surface of tissue culture dishes in isotropic conditions. However, when sarcoma cell aggregates were plated onto underlying aligned fibroblasts, the sarcoma cells showed contact guidance, migrating along the long axes of fibroblasts. Simultaneously, suppression of migration normal to the axis of fibroblasts orientation was observed. The sarcoma cells displaced a few times faster on aligned fibroblasts than under isotropic conditions in control cultures. This fast displacement was found to result from the less frequent cell turnings and straightening of cell trajectories (i.e., from klinokinesis), and not from an acceleration of cell movement and the longer cell tracks (i.e., not from orthokinesis). The presented results support the suggestion of Abercrombie (M. Abercrombie. 1979. Nature (London). 281: 259-262.) that tumour cells may be guided by the underlying normal cells when invading surrounding tissues and forming metastases.

Animals↗

PubMedDate not supplied

Noninvasive evaluation of immunosuppressive drug efficacy on acute donor cell survival.

PURPOSE: The therapeutic benefits of cell transplantation may depend on the survival of sufficient numbers of grafted cells. We evaluate four potent immunosuppressive medications aimed at preventing acute donor cell death. PROCEDURES AND RESULTS: Embryonic rat H9c2 myoblasts were stably transduced to express firefly luciferase reporter gene (H9c2-Fluc). H9c2-Fluc cells (3x10(6)) were injected into thigh muscles of Sprague-Dawley rats (N=30) treated with cyclosporine, dexamethasone, mycophenolate mofetil, tacrolimus, or saline from day -3 to day +14. Longitudinal optical bioluminescence imaging was performed over two weeks. Fluc activity was 40.0+/-12.1% (dexamethasone), 30.5+/-12.5% (tacrolimus), and 21.5+/-3.5% (mycophenolate) vs. 12.0+/-5.0% (control) and 8.3+/-5.0% (cyclosporine) at day 4 (P<0.05). However, by day 14, cell signals had decreased drastically to <10% for all groups despite drug therapy. CONCLUSION: This study demonstrates the ability of optical molecular imaging for tracking cell survival noninvasively and raises important questions with regard to the overall efficacy of immunosuppressives for prolonging transplanted cell survival.

Animals↗