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At least 343 records · Page 19Linked to original sources
PubMed1997

Determination of bacterial cell surface hydrophobicity of single cells in cultures and in wastewater in situ.

Bacterial cell surface hydrophobicity is one of the most important factors that influence bacterial adhesion. A new method microsphere adhesion to cells, for measuring bacterial cell surface hydrophobicity was developed. Microsphere adhesion to cells is based on microscopic enumeration of hydrophobic, fluorescent microspheres attaching to the bacterial surface. Cell surface hydrophobicity estimated by microsphere adhesion to cells correlates well with adhesion of bacteria to hydrocarbons or hydrophobic interaction chromatography for a set of hydrophilic and hydrophobic bacteria (linear correlation coefficients, R2, were 0.845 and 0.981 respectively). We also used microsphere adhesion to cells to investigate the in situ properties of individual free-living bacteria directly in activated sludge. Results showed that the majority of the bacteria were hydrophilic, indicating the importance of cell surface hydrophobicity for bacterial adhesion in sludge, and for the overall success of the wastewater treatment process.

Bacterial Adhesion↗
PubMed1987

Direct measurement of cell detachment force on single cells using a new electromechanical method.

We describe a new device in which an accurately measured force is applied to individual adherent cells while the topography of the adhesion zone is simultaneously monitored. The force is applied via a flexible glass micropipette, attached by suction to the cell under study, and is calculated directly from the measured pipette deflection. Regions of close contact in the adhesion zone are observed using interference reflection microscopy. We have used the device to measure the force required to detach human red blood cells from hydrophobic and hydrophilic glass surfaces, and to detach Dictyostelium discoideum amoebae from a hydrophobic glass surface. The measured forces per unit length of contact perimeter are within an order of magnitude of the tensions required for membrane rupture.

Cell Adhesion↗
PubMed2001

The process of cell adhesion among dissociated single cells of Hydra: morphological observations.

Ultrastructural observations were made on the initial adhesion process at the adherent region of Hydra endodermal cell pairs brought into contact (following dissociation) using a three-dimensional laser manipulator. Total contact length across the diameter of the adherent region decreased during the period 10-60 min after initial adhesion. However, the mean numbers of closest (<4 nm) and medium (5-25 nm) separation distances between membranes (thought to be important in total cell adhesion) were not significantly different. These data indicate that adherent cell pairs maintain a constant adhesiveness during the first 60 min of the adhesion process, despite membrane rearrangements. The relative length of each separation distance in adherent cell pairs approached that reported previously for intact Hydra. The sums of lengths in both the closest and medium categories (as a proportion of total contact length) increased because the length of cleavages (distances >25 nm) decreased significantly during the same time period. These results suggest that adherent cell pairs undergo rapid, active membrane changes in the adherent region, which might be associated with cell sorting. The possible significance of these changes for active rearrangement are discussed.

Animals↗
PubMed2004

DNA damage evaluation of hydroxyapatite on fibroblast cell L929 using the single cell gel electrophoresis assay.

Hydroxyapatite is the main component of the bone which is a potential biomaterial substance that can be applied in orthopaedics. In this study, the biocompatibility of this biomaterial was assessed using an in vitro technique. The cytotoxicity and genotoxicity effect of HA2 and HA3 against L929 fibroblast cell was evaluated using the MTT Assay and Alkaline Comet Assay respectively. Both HA2 and HA3 compound showed low cytotoxicity effect as determined using MTT Assay. Cells viability following 72 hours incubation at maximum concentration of both HA2 and HA3 (200 mg/ml) were 75.3 +/- 8.8% and 86.7 +/- 13.1% respectively. However, the cytotoxicity effect of ZnSO4.7H2O as a positive control showed an IC50 values of 46 mg/ml (160 microM). On the other hand, both HA2 and HA3 compound showed a slight genotoxicity effect as determined using the Alkaline Comet Assay following incubation at the concentration 200 mg/ml for 72 hours. This assay has been widely used in genetic toxicology to detect DNA strand breaks and alkali-labile site. The percentage of the cells with DNA damage for both substance was 27.7 +/- 1.3% and 15.6 +/- 1.0% for HA2 and HA3 respectively. Incubation of the cells for 24 hours with 38 microg/ml (IC25) of positive control showed an increase in percentage of cells with DNA damage (67.5 +/- 0.7%). In conclusion, our study indicated that both hydroxyapatite compounds showed a good biocompatibility in fibroblast cells.

Animals↗
PubMed1999

A single cell analysis of TCR AV24AJ18+ DN T cells.

The T-cell receptor (TCR) BV gene of human TCR AV24+ double-negative (DN) T cells, a novel subset of natural killer (NK) T cells, was investigated by single-cell sorting and single-cell polymerase chain reaction (PCR) methods. Seven of eleven TCR AV24+ DN T-cell clones utilized TCR BV8, three BV9, and one BV6. Six of seven TCR AV24/BV8+ DN T-cell clones had identical TCR beta and alpha chains, indicating that they were the same clone. All three TCR AV24/BV9+ DN T-cell clones also demonstrated the same amino acids in the CDR3 region. These findings strongly suggest that the usage of TCR beta and alpha chains on TCR AV24+ DN T cells is extremely restricted, supporting the notion that these cells recognize highly limited T-cell epitopes on antigens. All TCR AV24+ clones expressed the NKR-P1A mRNA, and so were true NK T cells. IL-2 and IL-4 mRNAs were detected in all clones, suggesting that the majority of these cells were Th0-type T cells. Six clones overexpressed Fas-ligand (Fas-L) mRNA and Fas antigen was detected on all clones at the mRNA level. In conclusion, TCR AV24+ DN T cells might recognize restricted T-cell epitopes on antigens and function as Th0-type T cells, inducer cells to Th1- or Th2-type T cells (regulatory T cells), and as Fas-L-positive cytolytic T cells.

Amino Acid Sequence↗
PubMed1996

[Studies on the phenotype deviation of the single-cell subclones of human stomach carcinoma MGC-803 cell line].

The diversity of phenotypes of tumor cells is the main reason for the differences of drug-sensitivity, immunology, metastatic properties and cellular growth rate, and thus raises difficulties in tumor diagnosis and therapy. In order to investigate the mechanisms involved in the expression, correlation, development and regulation of the diversed malignant phenotypes, cell models from single-cell subclones of human stomach carcinoma cell line MGC-803 were studied. According to the growing time needed for the process of separation of each single-cell subclone, they were categoried into 3 groups as fast growing, moderatly and slowly growing subclones and named. From each group, representative subclones were selected--M17 fast. M6 moderate and M3 slow, for further comparative studies. Foci frequency in soft agar was very high with M17, very low with M3, and M6, the middle. The difference in the major transformed phenotype among the 3 subclones were statistically significant. There were differences in the function of gap junctional intercellular communication as detected by scrape-loading and dye transfer method. M17 negative, while M6 and M3 were positive with dye transfer. Immunocytochemical staining of cytoskeleton elements showed heavy disruption of microtubule and microfilament networks and appearance of F-actin aggregates in M17 cells. On the other hand, M3 showed less disruptive changes and more normal appearance of microtubules and microfilament organization. Immunofluorescent staining showed marked differences in gene expression of c-myc, c-Ha-ras and c-met between M17 and M3. M17 expressed intense fluorescence of the 3 onco-proteins while M3 was negative with c-myc and c-Ha-ras, only weak c-met fluorescence in the cytoplasm.

Adenocarcinoma, Mucinous↗
PubMed2026

Network methods for diagonal integration of unpaired single-cell multiomics data: a review.

MOTIVATION: Advances in single-cell sequencing have enabled multiomics profiling at unprecedented resolution; however, mass spectrometry-based single-cell proteomics (scMS) remains inherently destructive, precluding simultaneous transcriptomic capture. Unlike antibody-based methods such as CITE-seq, which permit paired profiling but are restricted to targeted protein panels, scMS provides unbiased, genome-scale coverage of the intracellular proteome yet necessitates post hoc integration of unpaired datasets. This diagonal integration challenge, where transcriptomes and proteomes are measured in separate cells lacking shared anchors, remains underserved by existing reviews, which focus predominantly on vertical integration strategies enabled by non-destructive assays. RESULTS: We survey the complete computational pipeline for constructing mechanistic proteogenomic networks from unpaired single-cell data, covering: (i) unimodal network inference such as knowledge-based approaches, probabilistic graphical models, temporal directionality inference, and generative and foundation model strategies that establish the transcriptomic scaffold; (ii) cross-modal integration architectures such as network propagation, graph neural networks (scMRDR, scmFormer, scCotag), and consensus frameworks designed explicitly for the unpaired proteomics setting; and (iii) benchmarking paradigms spanning network reconstruction (BEELINE, GRETA, CausalBench) and multi-task integration evaluation (scMultiBench, SCMMIB), with guidance on metric selection under network sparsity and class imbalance. We identify three principal axes of future development: generative proteomic translation from transcriptomic precursors, inductive prior embedding in next-generation architectures, and perturbation-based causal benchmarking. AVAILABILITY AND IMPLEMENTATION: This is a review article; no novel software is distributed. A curated benchmark resource table, methods starter guide, and per-method bottleneck annotations are provided in the Supplementary Material.

Multiomics↗
PubMed2005

Single-cell microarray for analyzing cellular response.

Detection of cellular response by measuring intracellular calcium, (Ca2+)i with Ca2+-dependent fluorescent dye are standard approaches to detect ligand-stimulated cells and to study signaling through ligand/receptor interaction. We describe a single-cell microarray system to analyze cellular response of individual cells such as lymphocytes using microchamber array chips. The single-cell microarray chip is made from polystyrene with over 30,000 microchambers, which can accommodate only single cells. Lymphocytes derived from mouse spleen or human blood were spread on the microarray, and over 80% of the microchambers achieved single-cell status. Stimulation of B-cells through antigen receptors on the microarray allowed us to detect activated B-cells by comparing the states of single B-cells before and after stimulation with antigen, which is disabled for flow cytometry. In addition, this novel method demonstrated retrieval of positive single B-cells from microchambers by a micromanipulator and achieved antibody DNA analysis. The system is suitable for high-throughput analysis of intracellular Ca2+ response at the single-cell level and is applicable to screen antigen-specific lymphocytes for making specific monoclonal antibody.

Amino Acid Sequence↗
PubMed1994

Analysis of the B-cell progenitor compartment at the level of single cells.

BACKGROUND: During B-cell development in the mouse, the VH, DH and JH elements of the immunoglobulin heavy chain (IgH) locus are rearranged, firstly by DH-JH joining, and then by VH-DHJH joining. In-frame ('productive') VHDHJH joints and DHJH joints in reading frame 2 (one of the three possible DH reading frames) allow the expression of mu and truncated mu chains (D mu proteins), respectively. The expression of such molecules from one of the two IgH loci of a cell is thought to interfere with VH-DHJH recombination on the other IgH locus, and to guide the cells through further development. RESULTS: We have developed a gene amplification assay that permits the examination of rearranged immunoglobulin genes in single cells. Using this assay, we monitored cells bearing DHJH and/or VHDHJH joints at early stages of development: in CD43+ B-cell progenitors, subdivided into fractions A, B, C and C' by flow cytometry, and in CD43- pre-B cells (fraction D). Fraction C was enriched for cells with two non-productive VHDHJH joints. Cells containing both a DHJH joint in DH reading frame 2 and a VHDHJH joint were not seen in any fraction. All fraction D cells harbored an in-frame VHDHJH joint. Cells with two productive VHDHJH joints appear to be selected against throughout development. CONCLUSIONS: Cells expressing D mu proteins appear to be arrested in development as a result of inhibited VH-DHJH joining. Expression of the mu chain is required for maturation into CD43- pre-B cells; accordingly, cells carrying two non-productive VHDHJH joints accumulate in the CD43+ compartment. Such a developmental arrest may also affect cells that express self-reactive VHDHJH antibody domains. Our results indicate further that allelic exclusion at the IgH locus is already established at the pre-B cell stage.

Animals↗
PubMed2000

Single cell sorting and cloning.

Cell sorters now allow the selection of cells and other bodies according to a range of quite diverse criteria. The additional refinement that allows the sorting of individual cells based on these criteria has seen application in many fields of research. Single cells may be sorted for microscopy, for culture and for genetic analysis by way of single cell PCR (polymerase chain reaction). In practical terms, in the setting up of an instrument for single cell sorting, there are additional requirements to ensure that each detected event is indeed a single cell or body, that this cell can be reliably sorted via saline droplet, separate from its fellow travelers, that the aiming of the droplet deflection is sufficiently precise to find the target vessel and that the cell will be undamaged on arrival. Among the diverse reported applications of the technique, two fields which have benefited greatly are lymphocyte development and haemopoiesis. In the former case, the analysis of gene rearrangements in lymphocytes, both in the pre- and post-antigenic phases of development, has been enabled by the combined technologies of single cell sorting and PCR. It is argued that such experiments could not have been done without that partnership. In a similar way, the single cell sorting technique has been found to be the perfect way to demonstrate precursor/progeny relationships between haemopoietic cells and, further, to demonstrate rigorously the effects of particular cytokines on the haemopoietic system.

Cell Separation↗
PubMed2005

Microfluidic device for electric field-driven single-cell capture and activation.

A microchip that performs directed capture and chemical activation of surface-modified single cells has been developed. The cell capture system is comprised of interdigitated gold electrodes microfabricated on a glass substrate within PDMS channels. The cell surface is labeled with thiol functional groups using endogenous RGD receptors, and adhesion to exposed gold pads on the electrodes is directed by applying a driving electric potential. Multiple cell types can thus be sequentially and selectively captured on desired electrodes. Single-cell capture efficiency is optimized by varying the duration of field application. Maximum single-cell capture is attained for the 10-min trial, with 63 +/- 9% (n = 30) of the electrode pad rows having a single cell. In activation studies, single M1WT3 CHO cells loaded with the calcium-sensitive dye fluo-4 AM were captured; exposure to the muscarinic agonist carbachol increased the fluorescence to 220 +/- 74% (n = 79) of the original intensity. These results demonstrate the ability to direct the adhesion of selected living single cells on electrodes in a microfluidic device and to analyze their response to chemical stimuli.

Animals↗
PubMed2005

High-throughput single-cell fluorescence spectroscopy.

A high-throughput method for measuring single-cell fluorescence spectra is presented. Upon excitation with a 488 nm argon-ion laser many bacterial cells were imaged by a 20x microscope objective while they moved through a capillary tube. Fluorescence was dispersed by a transmission diffraction grating, and an intensified charge-coupled device (ICCD) camera simultaneously recorded the zero and the first orders of the fluorescence from each cell. Single-cell fluorescence spectra were reconstructed from the distance between zero-order and first-order maxima as well as the length and the pixel intensity distribution of the first-order images. By using this approach, the emission spectrum of E. coli cells expressing green fluorescent protein (GFP) was reconstructed. Also, fluorescence spectra of E. coli cells expressing non-fluorescent apo-subunits of R-phycoerythrin (R-PE) were recorded after incubation of the cells with phycoerythrobilin (PEB) chromophore. The fluorescence spectra are in good agreement with results obtained on the same cells using a fluorescence spectrometer or a fluorescence microscope. When spectra are to be acquired, this approach has a higher throughput, better sensitivity, and better spectral resolution compared to flow cytometry.

Bacterial Proteins↗
PubMed1985

Identification and Isolation of Single Cells that Produce Somatic Embryos at a High Frequency in a Carrot Suspension Culture.

A system was established in which single cells differentiated to embryos at a high frequency. Small spherical single cells from a carrot (Daucus carota L. cv "Kurodagosun") cell suspension culture were obtained by fractionation through sieving, using nylon screens and then density gradient centrifugation in Percoll solutions. Eighty-five to 90% of these small single cells differentiated to embryos when they were cultured in a medium containing 2,4-dichlorophenoxyacetic acid (5 x 10(-8) molar), zeatin (10(-6) molar), and mannitol (0.2 molar) for 7 days, followed by transfer to a medium containing zeatin (10(-7) molar) but no auxin. This indicates that there are at least two phases in the differentiation of embryos from single cells. The progression of the first phase required exogenous auxin, whereas that of the second phase was inhibited by the same growth regulator. The relationship between the morphology and potency for embryogenesis from single cells was discussed. The system established here is a useful one for investigation of differentiation process from a single cell to a whole plant via embryogenesis, especially in its early stage.

Journal Article↗
PubMed2006

High resolution array-CGH analysis of single cells.

Heterogeneity in the genome copy number of tissues is of particular importance in solid tumor biology. Furthermore, many clinical applications such as pre-implantation and non-invasive prenatal diagnosis would benefit from the ability to characterize individual single cells. As the amount of DNA from single cells is so small, several PCR protocols have been developed in an attempt to achieve unbiased amplification. Many of these approaches are suitable for subsequent cytogenetic analyses using conventional methodologies such as comparative genomic hybridization (CGH) to metaphase spreads. However, attempts to harness array-CGH for single-cell analysis to provide improved resolution have been disappointing. Here we describe a strategy that combines single-cell amplification using GenomePlex library technology (GenomePlex) Single Cell Whole Genome Amplification Kit, Sigma-Aldrich, UK) and detailed analysis of genomic copy number changes by high-resolution array-CGH. We show that single copy changes as small as 8.3 Mb in single cells are detected reliably with single cells derived from various tumor cell lines as well as patients presenting with trisomy 21 and Prader-Willi syndrome. Our results demonstrate the potential of this technology for studies of tumor biology and for clinical diagnostics.

Carcinoma, Renal Cell↗
PubMed1986

Mechanism of action of acetylcholine on calcium current in single cells from frog ventricle.

Ca currents (ICa) were measured by whole-cell patch clamp in single cells isolated from frog ventricle in which K currents were blocked with intracellular (120 mM) and extracellular (20 mM) Cs. Inward currents elicited by depolarizing voltage steps from a holding potential of -80 mV were blocked completely by 0.5 mM-Cd. The quality of the voltage clamp was assessed using two patch electrodes on a single cell. One electrode was used in the voltage-clamp mode to measure membrane currents and the other in current-clamp to measure membrane potential. Ca currents as large as 2 nA were well clamped in cells as long as 210 micron. Acetylcholine (ACh) had no effect on ICa in the absence of beta-adrenergic stimulation but reduced to control levels ICa elevated by isoprenaline. Nanomolar concentrations of ACh were able to reduce significantly ICa elevated by 2 microM-isoprenaline. ACh had no effect on the shape of the I-V curve, on the reactivation (recovery from inactivation), or on the inactivation of ICa. Although isoprenaline increased ICa by an average of 6.5-fold, it had no effect on the shape of the I-V curve or on the inactivation at test potentials negative to +40 mV. However, isoprenaline slowed the half-reactivation time from a control value of 120 +/- 10 ms (mean +/- S.D.) to 153 +/- 12 ms at -80 mV. The effect of cyclic AMP on ICa was investigated using two patch electrodes, one filled with cyclic AMP. Maximal effects of cyclic AMP were observed with 5 microM-cyclic AMP in the pipette. Maximal ICa was recorded several minutes after breaking the patch with the second electrode. After removing the cyclic-AMP-containing electrode, ICa declined to control levels after approximately 10 min. 5 microM-cyclic AMP in the patch electrode increased ICa by an average of 6.9-fold, but had no effect on the shape of the I-V curve or on inactivation. Cyclic AMP had a slowing effect on reactivation (half-reactivation time = 155 +/- 24 ms) similar to that of isoprenaline. ACh (1-10 microM) did not reduce ICa elevated with cyclic AMP (0.1-20 microM-cyclic AMP in the pipette). With low concentrations of cyclic AMP in the pipette (0.1 microM), isoprenaline augmented ICa, but with 5 microM-cyclic AMP in the pipette, isoprenaline was incapable of increasing ICa further. These results suggest that the decrease of ICa produced by ACh can be explained solely by decreases in cyclic AMP levels.

Acetylcholine↗