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Results for “single cell analysis”

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At least 127 records · Page 7Linked to original sources
PubMed1995

Use of 2,3-naphthalenedicarboxaldehyde derivatization for single-cell analysis of glutathione by capillary electrophoresis and histochemical localization by fluorescence microscopy.

We report that 2,3-naphthalenedicarboxaldehyde reacts rapidly with glutathione and its precursor, gamma-glutamylcysteine, to form highly fluorescent derivatives under physiological conditions. In contrast to previous accounts of 2,3-naphthalenedicarboxaldehyde labeling of primary amines, no additional CN- ion or any other additional nucleophile is required. The fluorescence spectral properties of the chromophores (lambda exc max = 472 nm, lambda em max = 528 nm) make these derivatives amenable to excitation and detection by optical instrumentation that is optimized for fluorescein wavelengths. This selective labeling chemistry enabled quantitative determination and histochemical localization of glutathione in neurobiological samples. Intracellular glutathione was labeled by incubating cultured cells or cell suspensions in a 2,3-naphthalenedicarboxaldehyde-supplemented, DMSO-containing physiological buffer (pH = 7.4) for 2-10 min. Applications include imaging of cultured NG 108-15 cells (mouse neuroblastoma x rat glioma) and primary glial and neuronal cell cocultures (rat hippocampus) using epiluminescent and confocal fluorescence microscopy. Quantitative determination of glutathione in single NG 108-15 cells was accomplished using laser-induced fluorescence detection and capillary electrophoresis.

Animals↗
PubMed2002

Single-cell analysis by a scanning thermal lens microscope with a microchip: direct monitoring of cytochrome c distribution during apoptosis process.

We developed a microsystem for cell experiments consisting of a scanning thermal lens microscope detection system and a cell culture microchip. The microchip system was good for liquid control in microspace, and this results in secure cell stimulation and coincident in vivo observation of the cell responses. The system could detect nonfluorescent biological substances with extremely high sensitivity without any labeling materials and had a high spatial resolution of approximately 1 microm. This system was applied to monitoring of cytochrome c distribution in a neuroblastoma-glioma hybrid cell cultured in the microflask (1 mm x 10 mm x 0.1 mm; 1 microL) fabricated in a glass microchip. Cytochrome c release from mitochondria to cytosol during the apoptosis process was successfully monitored with this system. The cytochrome c detected with this system was estimated to be approximately 10 zmol. We concluded that the system was suitable for measuring the distribution of chemical substances in a single cell because the microchip is good for liquid handling in microspace and the thermal lens microscope has high sensitivity and spatial resolution.

Apoptosis↗
PubMed1996

Single-cell analysis of T-cell receptor-gamma rearrangements in large-cell anaplastic lymphoma.

Large-cell anaplastic lymphomas (LCAL) are characterized by their distinctive morphology together with expression of the CD30 antigen. In addition, a chromosomal translocation, t(2;5) (p23; q35), can be detected in most cases. A significant proportion of LCALs carry rearrangements of the T-cell receptor-gamma (TCR-gamma) locus and display a T-cell phenotype. In about a third of the cases, another type of non-Hodgkin-lymphoma precedes LCAL. Early transformations of non-Hodgkin's lymphoma into LCAL might escape clinical detection in a significant number of cases. The existence of clonally related lymphoid cells within the lymph node infiltrates must be claimed in these cases. Recently, a small-cell-predominant variant of LCAL was described in which only few large tumor cells expressing the CD30 antigen are found together with numerous small lymphocytes, which are frequently CD30-. This observation in particular prompted us to investigate the clonal relationship of the tumor cell compartment and admixed small lymphocytes in one case of common LCAL with T-cell genotype. For this purpose, we chose to amplify rearranged TCR-gamma sequences from single cells isolated from immunostained frozen sections by using a micromanipulator. A total of 119 cells were investigated. Amplification products were obtained in 17 of 79 CD3+ cells, 12 of 30 CD30+ cells, and three of 10 CD20+ cells. The nucleotide sequences were determined in 28 cells by nonradioactive sequencing. In 11 CD30+ cells, the predominant rearrangement of TCR-gamma was identified. No clonal diversity was observed. The small CD3+ lymphocytes were unrelated to the anaplastic CD30+ tumor cells. This report describes a method to analyze rearrangements of the TCR-gamma in single cells isolated from immunostained frozen sections. Application of this technique revealed an absence of clonal diversity in a case of LCAL and documented the polyclonal nature of admixed small CD3+ lymphocytes.

Cell Separation↗
PubMed1996

Single cell analysis of H/RS cells.

The nature of the Hodgkin/Reed-Sternberg (H/RS) cells in Hodgkin's disease (HD) is still unclear. We have studied isolated H/RS cells from four cases of lymphocytic predominance (LP) HD and six cases of nodular sclerosis (NS) HD and assayed the cells for immunoglobulin heavy chain (IgH) gene rearrangement by the polymerase chain reaction (PCR). The H/RS cells in all four cases of LPHD had rearranged their IgH gene in a polyclonal pattern. The H/RS cells of three cases of NSHD where at least a fraction of the cells expressed the B-cell marker CD20 had also rearranged their IgH gene. In two of the cases, the rearrangements were totally unrelated while in one case, two out of six rearrangements were identical in sequence indicating the presence of a clonal subpopulation amid a polyclonal background. Clonality was assessed independently in one NSHD case by determining the pattern of X-chromosome inactivation. This technique also failed to show that the H/RS cells were monoclonal. Our studies suggest that in both LPHD and NSHD, the H/RS cells may be a polyclonal proliferation at presentation and clonal populations may emerge from this polyclonal background. There is also some evidence of a correlation between the immunophenotype and the genotype of the H/RS cells. Single cell assays show great potential in elucidating the cell lineage and clonality of H/RS cells, as well as their in vivo evolution during the disease process.

Gene Rearrangement↗
PubMed1997

Single cell analysis reveals regulated hierarchical T cell antigen receptor signaling thresholds and intraclonal heterogeneity for individual cytokine responses of CD4+ T cells.

T cell receptor (TCR) recognition of peptide-major histocompatibility complex antigens can elicit a diverse array of effector activities. Here we simultaneously analyze TCR engagement and the production of multiple cytokines by individual cells in a clonal Th1 CD4(+) cell population. Low concentrations of TCR ligand elicit only interferon-gamma (IFN-gamma) production. Increasing ligand recruits more cells into the IFN-gamma+ pool, increases IFN-gamma produced per cell, and also elicits IL-2, but only from cells already making IFN-gamma. Most cells producing only IFN-gamma show less TCR downmodulation than cells producing both cytokines, consistent with a requirement for more TCR signaling to elicit IL-2 than to evoke IFN-gamma synthesis. These studies emphasize the hierarchical organization of TCR signaling thresholds for induction of distinct cytokine responses, and demonstrate that this threshold phenomenon applies to individual cells. The existence of such thresholds suggests that antigen dose may dictate not only the extent, but also the quality of an immune response, by altering the ratios of the cytokines produced by activated T cells. The quantitative relationships in this response hierarchy change in response to costimulation through CD28 or LFA-1, as well as the differentiation state of the lymphocyte, explaining how variations in these parameters in the face of a fixed antigen load can qualitatively influence immune outcomes. Finally, although the IFN-gamma/IL-2 hierarchy is seen with most cells, among cells with the greatest TCR downmodulation, some produce only IFN-gamma and not IL-2, and the amount of IFN-gamma exceeds that in double producers. Thus, these single cell analyses also provide clear evidence of nonquantitative intraclonal heterogeneity in cytokine production by long-term Th1 cells, indicating additional complexity of T cell function during immune responses.

Amino Acid Sequence↗
PubMed1986

Single cell analysis of daunomycin uptake and efflux in multidrug-resistant and -sensitive KB cells: effects of verapamil and other drugs.

The accumulation of daunomycin in drug-sensitive and multidrug-resistant human KB cells was examined using light microscopy to detect the inherent fluorescence of daunomycin. Intracellular accumulation of fluorescent drug occurred rapidly in parental KB cells and was markedly reduced in several multidrug-resistant mutants. The addition of verapamil, which reverses multidrug resistance, resulted in increased accumulation of daunomycin in resistant cells. In living cells, most of the daunomycin was found in the nucleus, but significant amounts were detected associated with the plasma membrane, in the cytoplasm, in organelles of the Golgi region, and in lysosomes. The nuclear fluorescence was measured using a photometer system, and the loss of daunomycin from the cells was determined under various conditions. When sensitive cells were allowed to accumulate daunomycin for 5 min at 37 degrees C and then placed in medium without the drug, daunomycin remained inside the nuclei for longer than 1 day. When resistant cells were loaded in the presence of verapamil and the verapamil was removed, the resistant cells lost daunomycin with a half-time of about 1 min. The continuous presence of verapamil markedly inhibited the loss of daunomycin from the cells. Similar results were obtained in separate experiments using [3H]daunomycin. Vinblastine, vincristine, and quinidine were also effective in stimulating daunomycin accumulation in multidrug-resistant cells and in preventing the loss of daunomycin from these resistant cells. This effect required half-maximal concentrations of 1-2 microM for verapamil, vinblastine, and quinidine. Ouabain, lanthanum, colchicine, amiloride, probenecid, and 1-beta-D-arabinofuranosylcytosine had no effect on this process. Quinine was effective at 10 microM and nifedipine was effective at 50 microM. Depletion of cellular adenosine triphosphate levels by preincubation of cells with azide and 2-deoxyglucose partially inhibited daunomycin loss from resistant cells. These single-cell measurements indicate that diminished daunomycin accumulation in multidrug-resistant cells results from accelerated energy-dependent efflux across the plasma membrane, and this efflux is inhibited by verapamil, quinidine, vincristine, and vinblastine.

Biological Transport↗
PubMed2000

Single-cell analysis using capillary electrophoresis: influence of surface support properties on cell injection into the capillary.

Capillary electrophoresis (CE) is an important tool of chemical cytometry. Whole-cell analysis using CE starts with cell injection into the capillary by either siphoning or electroosmosis. However, strong adherence of the cell to the support surface can prevent efficient cell injection and lead to irreproducible analysis. Here we evaluated several surfaces as potential cell supports for HT29 cells (human colon adenocarcinoma). These cells strongly adhered to the surface of untreated glass or polystyrene. Hydrophobic coating with dimethyldichlorosilane (DMS) or Sigmacote did not significantly reduce cell adhesion. In contrast, cell adhesion was reduced significantly when the surface was modified with hydrophilic polymers (hydrogels) such as poly(2-hydrohyethyl methacrylate) (PHEMA) and polyvinyl alcohol (PVA). In addition to their pronounced antiadhesive properties, PHEMA and PVA coatings were the most biocompatible (had highest survival of cells in contact with surface). Hydrogel-coated polystyrene plates were tested as a commercial alternative to hydrogel-coated glass slides. The cell adhesive properties of such plates were similar to those of PHEMA and PVA. However, the biocompatibility of the plates was lower than that of the other surfaces tested. Moreover, in contrast to PHEMA- and PVA-coated glass slides, the plates were sensitive to UV light and therefore should not be used when fluorescent image microscopy with UV excitation precedes CE. The analyses of the data obtained showed that PHEMA- and PVA-coated glass slides were the most suitable cell supports for cell injection into the capillary.

Adenocarcinoma↗
PubMed2006

Single cell analysis of PKC activation during proliferation and apoptosis induced by laser irradiation.

Laser irradiation has been shown to trigger cellular proliferation and apoptosis in various cell types. Studying the signaling pathways involved in the laser irradiation is important for understanding these processes. In present study, to monitor the protein kinase Cs (PKCs) activity in living cells in real time, we transfected and screened human lung adenocarcinoma cells (ASTC-a-1) stably expressing C kinase activity reporter (CKAR) constructed based on fluorescence resonance energy transfer (FRET) technique. The CKAR is a specific, reversible reporter of phosphorylation by PKCs and it can monitor the ongoing balance between PKCs and phosphatases. The increasing dynamics of PKCs activity is monitored during cell proliferation induced by low-power laser irradiation (LPLI) (0.8 J/cm2) in serum-starved ASTC-a-1 cells stably expressing CKAR reporter using FRET imaging on laser scanning confocal microscope and using spectrofluorometric analysis on a luminescence spectrometer, respectively. However, the decreasing dynamics of PKCs activity has been monitored in real time using FRET imaging for the cells treated with high fluence LPLI (60 J/cm2), which was previously found to induce cell apoptosis. Taken together, LPLI induces the ASTC-a-1 cell proliferation by specifically activating PKCs. However, PKCs activity decreases during cell apoptosis induced by high fluence LPLI. Our results indicate that PKCs play an important role in the laser irradiation-induced biological effects.

Apoptosis↗
PubMed2003

Single-cell analysis reveals cell-specific patterns of expression of a family of putative voltage-gated sodium channel genes in the leech.

To understand the molecular basis of nervous system function in the leech, Hirudo medicinalis, we have isolated four novel cDNAs encoding putative voltage-gated sodium (Na) channel alpha subunits, and have analyzed the expression of these genes in individual neurons of known function. To begin, degenerate oligonucleotide primers were used in combination with pre-existing cDNA libraries and reverse transcriptase-coupled polymerase chain reactions (RT-PCR). The putative leech Na channel cDNAs (LeNas) exhibit a higher degree of sequence homology to Na channel genes in other species than to voltage-gated calcium or potassium channel genes, including those expressed in leech. All LeNa cDNAs contain sequences corresponding to regions of functional importance in Na channel alpha subunits, including the "S4 region" involved in activation, the "pore loops" responsible for ion selectivity, and the "inactivation loop" between the third and fourth domains, though the latter lacks the highly conserved "IFM" motif critical for mammalian Na channel inactivation. Sequences corresponding to important determinants of tetrodotoxin sensitivity are found in some, but not all, LeNa cDNAs, consistent with prior electrophysiological evidence of Na channel heterogeneity in the leech with respect to this toxin. Subsequently, two different sets of isoform-specific primers and methods of RT-PCR, including a sensitive, fluorescence-based "real time" RT-PCR, were used to analyze LeNa isoform expression in functionally distinct neurons. The results from both approaches were consistent, and not only demonstrated that individual neurons often express more than one LeNa isoform, but also revealed cell-specific patterns of Na channel isoform expression in the leech nervous system.

Amino Acid Sequence↗
PubMed2001

Graded mode of transcriptional induction in yeast pheromone signalling revealed by single-cell analysis.

Signalling pathways typically convert a graded, analogue signal into a binary cellular output. In the several eukaryotic systems that have been investigated to date, including MAP kinase cascade activation in Xenopus oocytes, analogue-to-digital conversion occurs at points in the pathway between receptor activation and the effector mechanism. We used flow cytometry combined with an intracellular fluorescent reporter to examine the characteristics of the yeast pheromone-response pathway. Surprisingly, pheromone response in yeast, which relies on the MAP kinase cascade, behaved in a fundamentally graded manner. Expression of certain exogenous dominant inhibitors of the pathway converted the response to graded-or-none behaviour. These results have implications for the dissection of biological response mechanisms in cells and illustrate how signalling pathways, even homologous ones, may have strikingly different signal propagation/amplification characteristics.

Flow Cytometry↗
PubMed1999

Single cell analysis of antigen-specific T cell responses in HIV-infected individuals.

Antigen-specific and polyclonally induced T cell responses were analyzed in 10 HIV-infected individuals and in 14 controls by enumerating the numbers of tetanus toxoid (TT)-specific and phytohemagglutinin (PHA)-induced IFN-gamma-secreting cells (SC) and IL-4-SC using an enzyme-linked immunospot assay. Whereas the numbers of IFN-gamma-SC in HIV-infected patients were not different from those of the controls in response to an in vitro stimulation with PHA, they were significantly decreased in response to an in vitro stimulation with TT, both before and after a TT booster. Cell depletion experiments indicated that this difference was related to an impairment of CD4(+) T-cell-mediated TT-specific IFN-gamma secretion. Concerning IL-4, the numbers of both polyclonally induced IL-4-SC and TT-specific IL-4-SC were significantly lower in HIV-infected patients than in the controls. It is concluded that secretion of antigen-specific cytokines of both Th1 and Th2 types is depressed in HIV-infected patients.

Adult↗
PubMed1992

Single cell analysis of factors increasing the survival of resealed erythrocytes in the circulation of mice.

Erythrocytes labeled with fluorescent membrane markers were reinfused into mice. Blood samples removed periodically were analyzed by flow cytometry for survival of labeled cells. Normal erythrocytes labeled with the markers persisted in the circulation for 45 days. A fraction of lysed and resealed erythrocytes labeled with marker were rapidly cleared from the circulation; the remainder were removed at approximately the same rate as normal cells in the same animal labeled with marker of another color. Lysis in a minimal volume of buffer and addition of ATP during lysis both increased the fraction of cells surviving initial clearance.

Adenosine Triphosphate↗
PubMed1993

Single cell analysis of the expression of a nuclear protein, SCIP, by fluorescent immunohistochemistry visualized with confocal microscopy.

A widely applicable method for the accurate quantification or semiquantification of macromolecules at the level of individual cells is described and validated; this is a method which may considerably facilitate the study of many biological processes. This method relies on measuring fluorescent emission in immunocytochemically labelled cells with a confocal microscope. Emission is related quantitatively to the level of the fluorophore by the combination of an analysis of the polarization of the fluorescent emission and fluorophore rationing methods. The method was applied to the study of the expression of the suppressed cyclic AMP-induced POU protein (SCIP) transcription factor in glial cells of the central nervous system. In particular, the method allowed the study of transcription factor expression in defined cells present in heterogeneous cultures and in cell types which cannot be isolated in sufficient numbers for biochemical analysis using conventional techniques.

Animals↗
PubMed1991

Single cell analysis in toxicity testing: the mitogenic activity of thioacetamide in cultured rat hepatocytes analyzed by DNA/protein flow cytometry.

Rat hepatocytes were cultured at 4% O2 and 13% O2 and exposed to the nongenotoxic rodent carcinogen thioacetamide (TA) from 24 to 72 h after isolation at exposure levels between 0.01 and 0.33 mM. Hepatocytes and isolated nuclei were analyzed by DNA-protein flow cytometry. An aggregate correction procedure was applied and the proportion of S-phase, diploid, tetraploid or octoploid hepatocytes as well as binucleated cells, were measured or calculated. The proportion of S-phase cells within the diploid hepatocytes increased with increasing concentration of TA up to 3.9-fold, whereas the corresponding increase in S-phase mononucleated tetraploid cells was only 1.8-fold. S-phase binucleate tetraploid cells showed no increase. In the tetraploid hepatocytes, the mitogenic stimuli was detectable only in cultures maintained at 4% O2. The relative contribution of binuclear cells was increased 1.5-fold in the octoploid cells. It is concluded that the mitogenic activity of TA initiates DNA synthesis in diploid hepatocytes in the G1 and in the following G2 cell-cycle phase, omitting karyogenesis. The cellular protein content is not affected which indicates that the mitogenic activity of the chemical is not necessarily associated with an increase in cellular protein content. The results obtained correspond well with data of in vivo studies. The method applied therefore allows the mitogenic activity of nongenotoxic carcinogens to be detected in vitro within 48 h and their mode of action to be elucidated.

Animals↗