Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Single cell”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 325 records · Page 18Linked to original sources
PubMed1994

Application of polymerase chain reaction (PCR) to the microscopically identified cells on the slides: evaluation of specificity and sensitivity of single cell PCR.

The sensitivity and specificity of single cell polymerase chain reaction (PCR) were studied. Its high sensitivity enabled detection of a single-copy gene, such as human T-lymphotropic virus type I genome in paraffin sections. The rate of obtaining positive signals with this method was affected by the number of copies of the gene in the target cell. Specificity was satisfactory if the procedure was properly and carefully followed. Since the single cell PCR is a time-consuming method which requires skill and experience to pick up the target cells accurately, the applicability of this method is limited. It works best when it is used to analyze a single or a few copy genes in histologically identified cells.

Blotting, Southern↗
PubMed1995

Efficiency of various dissociation methods for the preparation of thyroid single cell suspensions.

For comparison of the physiological potential of single thyroid cells versus cells integrated into follicles it would be ideal to work with suspensions consisting exclusively of single cells instead of a mixture of single cells and follicle fragments. In this study, various techniques for the isolation of single cells have been tested for their effect on cell viability, the ultrastructure of the isolated cells, the percentage of single cells and the ability of these cells to form follicles in culture. In addition, the cells were characterized for the preservation of their morphology and the ability to respond to TSH by comparing their immunocytochemical staining pattern with anti-vimentin and anti-ras p21 antibody to that of the intact thyroid tissue. Dispase treatment of thyroid tissues alone produced suspensions with a relatively small proportion of single cells. These cells stained with anti-vimentin and anti-ras p21 antibody to a similar percentage as thyroid cells in the intact gland. A combination of dispase treatment with either filtration or trypsin treatment severely compromised the viability of the cells. A high proportion of single cells with a good viability could be obtained either by centrifugation of dispase treated tissues or by culturing of dispase treated tissues as monolayers and subsequent detachment from the culture vessels with trypsin. Whereas the immunological staining with anti-vimentin and anti-ras oncogene antibody in the centrifuged cells resembled that of intact tissue, cells cultured as monolayers reacted differently. The differences in the immunological staining were still observed when the cells which had been grown as monolayers were stimulated with TSH. Differential centrifugation appeared to be the ideal method for the isolation of unaltered and viable single cells but is a rather laborious method to obtain larger amounts of single thyroid cells.

Animals↗
PubMed1995

Demonstration of chlorobenzene-induced DNA damage in mouse lymphocytes using the single cell gel electrophoresis assay.

The DNA damaging effect of chlorobenzene was investigated in peripheral lymphocytes and bone marrow cells from C57BL/6 female mice using a gel electrophoresis assay for DNA from single cells ('the single cell gel electrophoresis assay') under alkaline conditions. The effect of chlorobenzene was studied both after single and repeated intraperitoneal injections of 750 mg/kg body weight. The cytostatic agent cyclophosphamide (150 mg/kg, i.p.) was used as a reference substance, and vehicle-treated mice as controls. DNA damage was recorded 16 h after the (last) injection, using an automated computerized image analysis system specifically designed for the single cell gel electrophoresis assay. There was evidence of chlorobenzene-induced DNA damage after 3 days of repeated exposure in peripheral lymphocytes, but no indications of such an effect in bone marrow cells. Cyclophosphamide induced significant damage to DNA both in bone marrow cells and lymphocytes, the effect being most pronounced in the latter cells. It is concluded that high-dose exposure to chlorobenzene is associated with genotoxicity to peripheral lymphocytes. However, this solvent is apparently not a major hazard to bone marrow cells, even after repeated high-dose exposure.

Analysis of Variance↗
PubMed1990

Quantitative relationships between single-cell and cell-population model parameters for chemosensory migration responses of alveolar macrophages to C5a.

Phenomenological parameters from a mathematical model of cell motility are used to quantitatively characterize chemosensory migration responses of rat alveolar macrophages migrating to C5a in the linear under-agarose assay, simultaneously at the levels of both single cells and cell populations. This model provides theoretical relationships between single-cell and cell-population motility parameters. Our experiments offer a critical test of these theoretical linking relationships, by comparison of results obtained at the cell population level to results obtained at the single-cell level. Random motility of a cell population is characterized by the random motility coefficient, mu (analogous to a particle diffusion coefficient), whereas single-cell random motility is described by cell speed, s, and persistence time, P (related to the period of time that a cell moves in one direction before changing direction). Population chemotaxis is quantified by the chemotactic sensitivity, chi 0, which provides a measure of the minimum attractant gradient necessary to elicit a specified chemotactic response. Single-cell chemotaxis is characterized by the chemotactic index, CI, which ranges from 0 for purely random motility to 1 for perfectly directed motility. Measurements of cell number versus migration distance were analyzed in conjunction with the phenomenological model to determine the population parameters while paths of individual cells in the same experiment were analyzed in order to determine the single-cell parameters. The parameter mu shows a biphasic dependence on C5a concentration with a maximum of 1.9 x 10(-8) cm2/sec at 10(-11) M C5a and relative minima of 0.86 x 10(-8) cm2/sec at 10(-7) M C5a and 1.1 x 10(-8) cm2/sec in the absence of Ca; s and P remain fairly constant with C5a concentration, with s ranging from 2.1 to 2.5 microns/min and P varying from 22 to 32 min. chi 0 is equal to 1.0 x 10(-6) cm/receptor for all C5a concentrations tested, corresponding to 60% correct orientation for a difference of 500 bound C5a receptors across a 20 microns cell length. The maximum CI measured was 0.2. Values for the population parameters mu and chi 0 were calculated from single-cell parameter values using the aforementioned theoretical linking relationships. The values of mu and chi 0 calculated from single-cell parameters agreed with values of mu and chi 0 determined independently from population migrations, over the full range of C5a concentrations, confirming the validity of the linking equations. Experimental confirmation of such relationships between single-cell and cell-population parameters has not previously been reported.

Animals↗
PubMed2002

Negligible changes in piglet serum clinical indicators or organ weights due to dietary single-cell long-chain polyunsaturated oils.

Single-cell oils are currently included in human infant formula as sources of the long-chain polyunsaturates (LCP) docosahexaenoic acid (DHA) and arachidonic acid (AA) in many countries, but have not yet been approved for use in the USA. We prepared four bovine-milk-based formulas with AA/DHA=0, 34/17, 68/34 and 170/85 (mg per 100 kcal formula) provided by two commercial single-cell oils. These levels correspond approximately to 0, 1, 2 and 5 times the concentrations used in infant formulas and, due to greater consumption of formula per unit body weight, resulted in daily consumption of approximately 0, 3, 6 and 16 times those anticipated for human infants. All other dietary fat (47% of calories) was provided by a vegetable oil blend used in commercial human infant formulas. Domestic piglets were allowed to nurse with the sow for 24 h after parturition, then removed to individual cages and maintained on one of the four diets. At 30 days of age the piglets were sacrificed, and serum collected and organs weighed. With litters treated as a blocked variable, no significant differences among groups were found by analysis of variance for the following serum assays: alkaline phosphatase, alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), creatinine, albumin, glucose, cholesterol, triglycerides, and total protein. No significant differences were found for hematocrit or body weight. No significant differences were found among groups for weights of liver, brain, heart, lung, spleen, kidneys or lung, analyzed as absolute weight and as a fraction of body weight. Hematoxylin/eosin liver sections examined by light microscopy showed no abnormalities as evaluated by an independent pathologist. DHA content in liver and heart and AA content in heart showed significant dose-related accumulation (P<0.05) and confirmed enhanced tissue accretion of DHA and AA from both oils. We conclude that single-cell oils in formula consumed for 1 month in amounts up to 16-fold greater than proposed for human infants in the USA did not result in clinical chemistry or histopathologic indications of toxic effects in neonatal pigs.

Animals↗
PubMed2005

Multiple sampling in single-cell enzyme assays using CE-laser-induced fluorescence to monitor reaction progress.

A novel method for assaying enzymes from a single cell or small cell populations is described. The key advantage of this method is the ability to repeatedly sample a single cell enzyme reaction. Whereas multiple sampling has been achieved for larger cell types with a diameter of 1 mm, we report a technique by which single cell enzyme assays of small cells (15 microm in diameter) can be repeatedly carried out. Individual cells were isolated using an in-house-built micromanipulator and placed in nanoliter-scale reaction vessels. The cells were lysed with solution containing substrate, and enzyme activity was assayed by removing 5-nL aliquots with a recently developed nanopipettor. The reaction aliquot was then analyzed using capillary electrophoresis with laser-induced fluorescence detection to quantitate enzyme activity. Sf9 cells were assayed at the single cell level and found to be highly heterogeneous with respect to alpha-glucosidase II activity. Since only 5 nL of the single cell reaction was removed, multiple sampling was possible, allowing triplicate analysis of enzyme activity for each individual cell. Multiple sampling also permitted a single cell reaction to be monitored over time. The sensitivity of this method was demonstrated in the analysis of a low-abundance enzyme, alpha1,3-N-acetylgalactosaminyltransferase, from single HT29 cells. Detecting the product of this enzyme reaction required minimizing the dilution of cellular contents. To demonstrate the potential applications of this methodology in small scale biochemical analyses, single Arabidopsis knf embryos lacking the alpha-glucosidase I encoding KNOPF gene were assayed. Mutant embryos demonstrated insignificant conversion of a triglucose substrate, as compared to wild type, confirming the deletion of alpha-glucosidase I. Embryos were simultaneously assayed for a second enzyme, beta-galactosidase, illustrating that the mutants were viable except for their lack of alpha-glucosidase I activity.

Animals↗
PubMed1998

Detection of gene expression in single neurons by patch-clamp and single-cell reverse transcriptase polymerase chain reaction.

Detection and quantitation of gene expression in single cells is especially important in the central nervous system where, at the cellular level, the synapse can be considered the single functional unit. For example, the consolidation of long-term memories may be mediated by persistent changes in the strength of synaptic transmission at individual synapses. In order to investigate the requirement for de novo RNA synthesis during long-term potentiation in individual neurons, we have combined single-cell electrophysiology with single-cell gene-expression methodology. Described are methods combining whole-cell patch-clamp and single-cell RT-PCR for the detection of a single mRNA species for nitric oxide synthase, or, through a multiplex strategy, for the simultaneous detection of several mRNAs including heme oxygenase 2, protein phosphatase inhibitor 1 protein, and several isoforms of the calcium/calmodulin dependent protein kinase II.

Animals↗
PubMed1995

Enzymatic amplification of specific deoxyribonucleic acid sequences from single cells: evaluation of a simplified and rapid method for use in preimplantation genetic diagnosis.

OBJECTIVE: To develop a simplified polymerase chain reaction (PCR) protocol on single cells for the purpose of preimplantation genetic diagnosis. Also to evaluate a new thermal cycler, RoboCycler 40 (Stratagene, La Jolla, CA), for reducing the time to complete PCR amplification. DESIGN: PCR amplification without DNA purification or reamplification of a 149 base pair (bp) segment of the human Y chromosome was used as a model. The assay was tested in human fetal cells, single lymphocytes and single human blastomeres. RESULTS: Amplification of the 149 bp segment using fetal cells was 100% correct. Results on single lymphocytes were concordant in all but one of the 15 male cases. However, 2 of the 25 female cases were identified as male suggesting the occurrence of DNA contamination. Analysis of 61 blastomeres were concordant in 57 cases (93%); results for male blastomeres showed 12% of false negatives. No false positives were detected for female cells. Amplification using the simplified PCR protocol in combination with the RoboCycler was completed in 2 hours. CONCLUSION: These data show that this PCR assay performed directly, without DNA extraction or purification and without re-amplification is a practical and effective approach for amplification of specific DNA sequences in single cells. Furthermore, the simplified PCR protocol significantly reduced the time to complete DNA amplification. The reduced time is expected to facilitate the management of a routine program for preimplantation genetic diagnosis.

Base Sequence↗
PubMed1999

Identification of ataxia telangiectasia heterozygotes, a cancer-prone population, using the single-cell gel electrophoresis (Comet) assay.

Heterozygotes of ataxia telangiectasia (AT) may comprise up to 1% of the general population. Because these individuals have no clinical expression of AT but may be highly radiosensitive and strongly predisposed for several forms of cancer, identification of AT carriers represents a considerable interest in cancer epidemiology and radiotherapy. We report a new approach for the in vitro identification of AT-heterozygotes based on the evaluation of the radiosensitivity and DNA damage repair ability of peripheral blood mononuclear cells using the single-cell gel electrophoresis (Comet) assay. The assay was performed on cells isolated from four different groups of individuals: (1) apparently healthy donors (n = 10); (2) patients with breast cancer showing a normal reaction to radiotherapy (n = 10); (3) a group of obligate AT carriers (parents of AT-homozygotes, n = 20); and (4) AT-homozygotes (n = 4). Cells irradiated with 3 Gy of x-rays were assayed for three parameters: (1) the initial and (2) residual DNA damage and (3) the kinetics of DNA damage repair. Both AT-heterozygotes' and AT-homozygotes' cells were found to be highly sensitive to x-irradiation. Quantitative evaluation of the single-cell electrophoregrams revealed that the average initial DNA damage in AT-heterozygous and AT-homozygous cells was almost three times higher than that in control non-AT cells. In addition, the DNA repair process in irradiated AT carrier cells was almost three times slower, and the extent of irreparable DNA damage in these cells was three times greater than in controls. Simultaneous assessment of the three parameters enabled correct identification of all tested AT carriers. This method seems to be a sensitive and useful tool for populational studies as a rapid prescreening test for a mutated AT status. The approach can also be extended for prediction of the in vivo radiosensitivity, which would enable optimization of individual radiotherapy schedules.

Adolescent↗
PubMed1998

T cell syncytia induced by HIV release. T cell chemoattractants: demonstration with a newly developed single cell chemotaxis chamber.

A chemotaxis chamber has been developed to analyze both the velocity and the directionality of individual T cells in gradients of high molecular mass molecules over long periods of time. Employing this chamber, it is demonstrated that syncytia induced by HIV in SUP-T1 cell cultures release two T cell chemoattractants with approximate molecular masses of 30 and 120 kDa. Neither uninfected single cells nor polyethylene glycol-induced syncytia release detectable chemoattractant, suggesting that these chemoattractants are linked to HIV infection. Soluble gp120 functions as a T cell chemoattractant and the addition of anti-gp120 antibody to syncytium-conditioned medium blocks the high molecular mass chemoattractant activity but not the low molecular mass activity. The addition of anti-CD4 antibody to syncytium-conditioned medium also blocks the high molecular mass chemoattractant activity but not the low molecular mass activity. These results demonstrate that HIV-induced T cell syncytia release a low and a high molecular mass T cell chemoattractant, and suggest that the high molecular mass factor is gp120 and that it functions through the CD4 receptor.

CD4 Antigens↗
PubMed2005

Analysis of FOXP3 protein expression in human CD4+CD25+ regulatory T cells at the single-cell level.

The transcription factor FOXP3 plays a key role in CD4(+)CD25(+) regulatory T cell function and represents a specific marker for these cells. Despite its strong association with regulatory T cell function, in humans little is known about the frequency of CD4(+)CD25(+) cells that express FOXP3 protein nor the distribution of these cells in vivo. Here we report the characterization of seven anti-FOXP3 monoclonal antibodies enabling the detection of endogenous human FOXP3 protein by flow cytometry and immunohistochemistry. Flow-cytometric analysis showed that FOXP3 was expressed by the majority of CD4(+)CD25(high) T cells in peripheral blood. By contrast, less than half of the CD4(+)CD25(int) population were FOXP3(+), providing an explanation for observations in human T cells that regulatory activity is enriched within the CD4(+)CD25(high) pool. Although FOXP3 expression was primarily restricted to CD4(+)CD25(+) cells, it was induced following activation of both CD4(+) and CD8(+) T cell clones. These findings indicate that the frequency of FOXP3(+) cells correlates with the level of expression of CD25 in naturally arising regulatory T cells and that FOXP3 protein is expressed by some activated CD4(+) and CD8(+) T cell clones. These reagents represent valuable research tools to further investigate FOXP3 function and are applicable for routine clinical use.

Animals↗
PubMed1999

The kinetics and stimulant dependence of cytokine production by blood and bronchoalveolar lavage T cells evaluated at the single cell level.

We have previously shown that bronchoalveolar lavage (BAL) T cells from human airways predominantly produce interferon gamma (IFN-gamma) and interleukin 2 (IL-2) when stimulated ex vivo. The kinetics of TH1 and TH2 cell cytokine production by T cells from both blood and BAL were studied to establish the optimal time after stimulation either with pharbol myristate (PMA) and ionomycin or with the more physiological stimulus of anti-CD3 for intracellular cytokine detection of IFN-gamma, IL-2, IL-4 and IL-5 in both blood and BAL T cells. The optimal time for positive identification of IL-2 in both blood and BAL was 5 h after PMA/ionomycin stimulation, whereas the first peak for IFN-gamma was found after 5 h in blood but after only 3 h in BAL. T cells from different biological compartments responded differently to each of the stimuli. Whilst anti-CD3 stimulation did not induce TH1 cytokine production in blood T cells, it readily induced both IFN-gamma and IL-2 production in BAL T cells. The kinetics of cytokine production were found to be stimulus dependent. Whilst IL-2 production showed similar kinetics with both stimuli, the kinetics of IFN-gamma production differed between stimuli. We have also examined the effect of five different stimuli on cytokine production by T cells to determine whether different forms of stimulation may selectively stimulate or inhibit different cytokines. Not surprisingly, PMA/ionomycin induced a greater percentage of BAL T cells to produce TH1 cytokines. However, other than modest amounts of the TH2 cytokines IL-4 and IL-5 were not induced by any of the five stimuli.

Adult↗
PubMed2006

High-resolution video monitoring of hematopoietic stem cells cultured in single-cell arrays identifies new features of self-renewal.

To search for new indicators of self-renewing hematopoietic stem cells (HSCs), highly purified populations were isolated from adult mouse marrow, micromanipulated into a specially designed microscopic array, and cultured for 4 days in 300 ng/ml Steel factor, 20 ng/ml IL-11, and 1 ng/ml flt3-ligand. During this period, each cell and its progeny were imaged at 3-min intervals by using digital time-lapse photography. Individual clones were then harvested and assayed for HSCs in mice by using a 4-month multilineage repopulation endpoint (>1% contribution to lymphoid and myeloid lineages). In a first experiment, 6 of 14 initial cells (43%) and 17 of 61 clones (28%) had HSC activity, demonstrating that HSC self-renewal divisions had occurred in vitro. Characteristics associated with HSC activity included longer cell-cycle times and the absence of uropodia on a majority of cells within the clone during the final 12 h of culture. Combining these criteria maximized the distinction of clones with HSC activity from those without and identified a subset of 27 of the 61 clones. These 27 clones included all 17 clones that had HSC activity; a detection efficiency of 63% (2.26 times more frequently than in the original group). The utility of these characteristics for discriminating HSC-containing clones was confirmed in two independent experiments where all HSC-containing clones were identified at a similar 2- to 3-fold-greater efficiency. These studies illustrate the potential of this monitoring system to detect new features of proliferating HSCs that are predictive of self-renewal divisions.

Animals↗
PubMed2006

Functional demonstration of the ability of a primary spermatogonium as a stem cell by tracing a single cell destiny in Xenopus laevis.

In Xenopus, although primary spermatogonium (PG), the largest cell in the testis, is believed to be spermatogonial stem cell by histological observations, functional evidence has never been obtained. In the present study, we first indicated that culture of juvenile testis in a medium supplemented with follicle stimulating hormone resulted in no proliferation of PG. In this culture system, early secondary spermatogonia could undergo mitotic divisions with a concomitant decrease in their size, so that they became distinguishable in size from PG. Because the subcutaneous environment of juveniles permitted aggregates of the dissociated testicular cells to reconstruct the normal testis structure, we next inserted a genetically marked PG isolated from cultured testes into the aggregate and transplanted it subcutaneously. In this system, 73.9% of the aggregates contained a marked PG. When we observed the aggregates 12 weeks after transplantation, most aggregates (70.0%) contained marked PG that had self-renewed. Among these, fully growing aggregates contained many spermatogenic cells at the later developmental stage. These results suggested that isolated PG from the cultured testes had the ability as stem cells, and that purification of the spermatogenic stem cells became reliable in Xenopus.

Animals↗
PubMed1992

Hydrogen Ion Regulation in Rat Cerebellar Granule Cells Studied by Single-Cell Fluorescence Microscopy.

The internal pH (pHi) regulatory mechanisms of individual rat cerebellar granule cells maintained in tissue culture have been investigated using the fluorescent indicator BCECF (2,7' bis carboxyethyl 5,6 carboxy-fluorescein) and quantitative fluorescence microscopy. The steady-state pHi was estimated as 7.27 +/- 0.25 in bicarbonate-buffered media and 7.49 +/- 0.35 in HEPES-buffered media. Buffering power was estimated at about 8 mM/pH unit from the peak alkalinization and acidification transients seen on addition and removal of NH4Cl. Bicarbonate did not appear to contribute to the buffering power estimated in this way. Following an acid load imposed by the ammonium prepulse technique, pHi recovered to steady-state values with first-order kinetics. Recovery was absolutely dependent upon extracellular sodium and, in about half of the cells tested, bicarbonate ions. In cells that did not require bicarbonate for pHi recovery, amiloride (1 mM) inhibited pHi recovery. Removal of extracellular chloride produced a reversible alkalinization of pHi in a third of the cells studied. This alkalinization persisted even when extracellular sodium had been reduced to zero. Removal of extracellular chloride did not inhibit bicarbonate-dependent pHi recovery following an acid load. These results are best explained by the existence of three independent pHi regulatory mechanisms: Na+/H+ exchange, Na+/HCO3- cotransport and Cl-/HCO3- exchange.

Journal Article↗