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At least 379 records · Page 21Linked to original sources
PubMed2004

The interferon gamma secretion assay: a reliable tool to study interferon gamma production at the single cell level.

Different single-cell analyses for the detection of antigen-specific T cells based on antigen-triggered induction of cytokine production (elispot, intracellular cytokine staining, cytokine secretion assay, etc.) have been analyzed. In this paper we present the data of a thorough validation of the IFNgamma Secretion Assay (ISA, Miltenyi Biotec, Bergisch Gladbach, Germany). In this assay the secreted IFNgamma is bound to the cell surface and is then stained as an artificial surface molecule and analyzed by flow-cytometry. The introduction of five quality criteria markedly improved the reproducibility of this assay and made it very reliable (intra-assay variability<5%; inter-assay variability<20%). Recovery experiments further demonstrated that almost 100% of IFNgamma(+) labeled cells could be detected by this technology. In order to analyze which cell subsets contribute to IFNgamma-production, we compared the results obtained in different individuals after VZAg-stimulation. Three different IFNgamma-secretion patterns could be discerned. In Pattern 1 there is a predominant and almost equal contribution of T cells and NK cells with a minor contribution of CD3(+)CD56(+) and B cells. Pattern 2, which is most abundant, is characterized by a predominance of NK cells (60-70%). Pattern 3 differs from the previous one in its minor contribution of NK cells. Here T cells predominate the IFNgamma secretion. These results clearly demonstrate that the IFNgamma(+) subset distribution after VZAg-stimulation is not uniform and differs individually. Furthermore, the ISA-technology proves to be very useful in vaccine research. This was demonstrated by testing the IFNgamma(+) secretion pattern after HBsAg-stimulation in PBMC from HBsAg-vaccinated individuals.

Antibodies, Viral↗
PubMed2005

Micromanipulation of single cells from tissue imprints is an alternative to laser-assisted microdissection.

The characterization and analysis of single cells by molecular biological methods such as the polymerase chain reaction (PCR) is of increasing interest in biomedical research. Different techniques have been developed to obtain single cells from solid tissue. Currently, the most frequently used technique is laser-assisted microdissection (LAM). However, LAM of tissues cannot exclude contamination of the targeted cells by underlying cell fragments. Moreover, this technique can only be performed if a laser microscope is available. Thus, we developed a method to obtain single cells of fresh solid tissue by the simple technique of tissue imprints. After immunostaining of the imprints, single cells were transferred to a reaction tube using a 27-gauge needle guided by a mechanical micromanipulator. Consequently, we used these cells in a single cell PCR.

Base Sequence↗
PubMed1999

Measurement of DNA cross-linking in patients on ifosfamide therapy using the single cell gel electrophoresis (comet) assay.

The single cell gel electrophoresis comet assay has become established as a sensitive technique for measuring DNA strand breaks. The technique has been modified to allow the sensitive detection and quantitation of DNA interstrand cross-linking at the single cell level. Cells are irradiated immediately before analysis to deliver a fixed level of random strand breakage. After embedding of cells in agarose and lysis, the presence of cross-links retards the electrophoretic mobility of the alkaline denatured cellular DNA. Cross-links are, therefore, quantitated as the decrease in the comet tail moment compared with irradiated controls. Using this method, a linear response of cross-linking versus dose of chlorambucil over a wide dose range was demonstrated in human lymphocytes after drug treatment ex vivo. The method was also sensitive enough to determine cross-linking in clinical samples after chemotherapy. For example, crosslinking was observed in the lymphocytes of patients receiving ifosfamide (3 g/m2/day) as a continuous infusion for 3-5 days or as a 3-h infusion daily for 3 days. Cross-links were detected in all patients within 3 h, with no evidence of DNA single strand break formation. In patients receiving continuous infusion, a plateau of cross-linking was reached by 24 h. In the patients receiving ifosfamide over 3 h, a clear decrease in the peak level of cross-linking was observed before subsequent infusions.

Antineoplastic Agents, Alkylating↗
PubMed1991

Chemiluminescence detection of proteins from single cells.

The analysis of proteins from single cells requires techniques of supreme sensitivity. Although radiochemical procedures are capable of detecting small amounts of electrophoretically separated proteins, their sensitivity falls short of that required for routine detection of minor components of single cells. Utilizing the avidin-biotin interaction and the alkaline phosphatase substrate 3-(4-methoxyspiro[1,2-dioxetane-3,2'- tricyclo-[3.3.1.1(3,7)]decan]-4-yl)phenyl phosphate (AMPPD), we have developed an alternative, chemiluminescence-based method for protein detection whose sensitivity exceeds that of other methods. Applying this method to a purified protein, we could detect as little as 63 fg (0.9 amol) of biotinylated bovine serum albumin. The sensitivity of the method was demonstrated by the detection of proteins from individual photoreceptor outer segments, including proteins constituting approximately 1% of the total. Chemiluminescence detection also proved extremely sensitive for immunoblotting: a comparison of five methods for detection of antibody-antigen interactions showed that the AMPPD technique was more sensitive than detection with a colorimetric alkaline phosphatase substrate, 125I-labeled protein A, 125I-labeled anti-mouse IgG, or colloidal gold-conjugated anti-mouse IgG.

Alkaline Phosphatase↗
PubMed2025

scMGCL: accurate and efficient integration representation of single-cell multi-omics data.

MOTIVATION: Single-cell multi-omics data integration is essential for understanding cellular states and disease mechanisms, yet integrating heterogeneous data modalities remains a challenge. We present scMGCL, a graph contrastive learning framework for robust integration of single-cell ATAC-seq and RNA-seq data. Our approach leverages self-supervised learning on cell-cell similarity graphs, in which each modality's graph structure serves as an augmentation for the other. This cross-modality contrastive paradigm enables the learning of biologically meaningful, shared representations while preserving modality-specific features. RESULTS: Benchmarking against state-of-the-art methods demonstrates that scMGCL outperforms others in cell-type clustering, label transfer accuracy, and preservation of marker-gene correlations. Additionally, scMGCL significantly improves computational efficiency, reducing runtime and memory usage. The method's effectiveness is further validated through extensive analyses of cell-type similarity and functional consistency, providing a powerful tool for multi-omics data exploration. AVAILABILITY AND IMPLEMENTATION: Code and datasets are released at https://github.com/zlCreator/scMGCL.

Single-Cell Analysis↗
PubMed1996

Morphogenesis in leaf and single-cell cultures of mature Juniperus oxycedrus.

Single cells were mechanically isolated from leaf-derived callus of mature Juniperus oxycedrus L. These cells divided and gave rise to callus when plated on medium containing growth regulators. Best plating efficiency was obtained on a modified Schenk and Hildebrandt medium supplemented with 0.6 micro M 2,4-dichlorophenoxyacetic acid and 100 mg l(-1) casein hydrolyzate. Although single-cell-derived callus showed poor morphogenic potential, both adventitious shoots and embryogenic tissues differentiated from the callus. We also achieved induction of somatic embryogenesis in leaf explants of mature J. oxycedrus trees cultured in the presence of 6.0 or 10.0 micro M 2,4-dichlorophenoxyacetic acid or picloram. Frequency of embryogenic callus ranged from 6 to 18%; however, under the culture conditions tested, isolated embryos failed to develop into plants.

Journal Article↗
PubMed2004

Establishment by an original single-cell cloning method and characterization of an immortal mouse melanoblast cell line (NCCmelb4).

We devised a unique new single-cell cloning method which uses microscope cover glasses and established a melanoblast cell line derived from mouse neural crest cells. A microscope cover glass was nicked and broken into small pieces and put on a dish. Culture medium and a suspension of 20-30 cells/ml were dropped in the dish. After 1-3 d, a piece of glass to which only one cell was adhered was picked up and transferred to another dish containing culture medium. The greatest advantage of this method is that the derivation of a colony from a single cell can be directly confirmed by microscopy and there is no risk of migratory cells being contaminated by other colonies. Using this single-cell cloning method, in this study we established a cell line derived from a neural crest cell line (NCC-S4.1) and designated it as NCCmelb4. When the culture medium was supplemented with stem cell factor (SCF) alone, NCCmelb4 cells were KIT-positive and tyrosinase-negative melanocyte precursors; they remained at an immature and undifferentiated stage. When the medium was supplemented with phorbol 12-o-tetradecanoyl-13-acetate (TPA) + cholera toxin (CT), the cell morphology changed and became L-3,4-dihydroxyphenylalanine (DOPA)-positive. This observation indicates that the NCCmelb4 cells are capable of further differentiation with suitable stimulation. NCCmelb4 cells derived from the mouse neural crest has characteristics of melanocyte precursors (melanoblasts), and is a cell line which can be utilized to study differentiation-inducing factors and growth factors without the effects of feeder cells.

Animals↗
PubMed2005

Connections between single-cell biomechanics and human disease states: gastrointestinal cancer and malaria.

We investigate connections between single-cell mechanical properties and subcellular structural reorganization from biochemical factors in the context of two distinctly different human diseases: gastrointestinal tumor and malaria. Although the cell lineages and the biochemical links to pathogenesis are vastly different in these two cases, we compare and contrast chemomechanical pathways whereby intracellular structural rearrangements lead to global changes in mechanical deformability of the cell. This single-cell biomechanical response, in turn, seems to mediate cell mobility and thereby facilitates disease progression in situations where the elastic modulus increases or decreases due to membrane or cytoskeleton reorganization. We first present new experiments on elastic response and energy dissipation under repeated tensile loading of epithelial pancreatic cancer cells in force- or displacement-control. Energy dissipation from repeated stretching significantly increases and the cell's elastic modulus decreases after treatment of Panc-1 pancreatic cancer cells with sphingosylphosphorylcholine (SPC), a bioactive lipid that influences cancer metastasis. When the cell is treated instead with lysophosphatidic acid, which facilitates actin stress fiber formation, neither energy dissipation nor modulus is noticeably affected. Integrating recent studies with our new observations, we ascribe these trends to possible SPC-induced reorganization primarily of keratin network to perinuclear region of cell; the intermediate filament fraction of the cytoskeleton thus appears to dominate deformability of the epithelial cell. Possible consequences of these results to cell mobility and cancer metastasis are postulated. We then turn attention to progressive changes in mechanical properties of the human red blood cell (RBC) infected with the malaria parasite Plasmodium falciparum. We present, for the first time, continuous force-displacement curves obtained from in-vitro deformation of RBC with optical tweezers for different intracellular developmental stages of parasite. The shear modulus of RBC is found to increase up to 10-fold during parasite development, which is a noticeably greater effect than that from prior estimates. By integrating our new experimental results with published literature on deformability of Plasmodium-harbouring RBC, we examine the biochemical conditions mediating increases or decreases in modulus, and their implications for disease progression. Some general perspectives on connections among structure, single-cell mechanical properties and biological responses associated with pathogenic processes are also provided in the context of the two diseases considered in this work.

Animals↗
PubMed1995

Simultaneous DNA 'fingerprinting', diagnosis of sex and single-gene defect status from single cells.

Sex and cystic fibrosis status have been previously diagnosed separately at the single cell level. We have developed a sensitive, reliable, accurate and rapid (within 5-6 h) system for the simultaneous diagnosis of sex, cystic fibrosis and a DNA 'fingerprint' within a single reaction from a variety of single cells. As contamination cannot be totally excluded, particularly at the single cell level, DNA 'fingerprinting' can be used to assess the risk of contamination. High sensitivity with single cells is combined with very high specificity (estimated matching probability of 10(-7)-10(-8)), allowing the source of the amplified cell to be identified with a very high degree of probability. Fluorescent primers were multiplexed for six tetranucleotide microsatellite sequences to determine the DNA fingerprint; the amelogenin gene was used to diagnose sex, and primers for the CFTR region were used to determine cystic fibrosis (CF) status. Analysis of the fluorescent product was undertaken using an automated DNA sequencer with Genescan software. This technique has many applications such as prenatal and preimplantation diagnosis, forensic identification of small or degraded samples, and detection of contamination sources. DNA fingerprints of single haploid spermatozoa and other cells can be assessed, so ensuring the detection of both diploid and haploid contamination during preimplantation diagnosis.

Cystic Fibrosis↗
PubMed1991

Immunofluorescence-digital image processing system for the quantitation of secreted immunoglobulin by single cells.

To quantitate the amount of secreted immunoglobulin (Ig) by a single cell, the immunofluorescence digital image processing (IDIP) system was adapted to the modified enzyme-linked immunospot (ELI-SPOT) assay. In this assay, an immunofluorescence (tetramethylrhodamine isothiocyanate) conjugated antibody was used for the detection of spots instead of the usual method of enzyme coupling. We have named this the immunofluorescence-linked immunospot (ILISPOT) assay. In addition to the quantitation of secreted Ig by single cells, this method allowed us to objectively determine the exact number of Ig producing spot forming cells (SFC). 96 well culture plates were pre-coated with goat anti-mouse Ig. The mouse IgM producing hybridoma (E-3-4) was incubated in the plates for 4 h at 37 degrees C. Cells were removed prior to the addition of biotinylated goat anti-mouse mu antibody. After overnight incubation, immunofluorescence conjugated avidin was added for the visualization of spots by the IDIP system. The IDIP system consists of a fluorescent microscope equipped with a video camera and computer. The gray scale of secreted IgM was initially established as a standard by the known amount of purified IgM. By using digital image processing, the number of spots and the gray scale of individual spots were computed. The shape and pattern of gray scale data were used to distinguish between the real spots and pseudo spots. This IDIP system could detect as little as 0.19 pg of secreted IgM (1.2 x 10(5) molecules) and an average of approximately 1.33 pg (8.3 x 10(5) molecules) produced by a single cell. Adaptation of the digital image processing system to the ILISPOT assay allowed the measurement of both the amount of Ig produced at the single cell level and also the exact numbers of SFC present in a totally objective fashion.

Animals↗
PubMed2005

High-throughput screening of enzyme libraries: thiolactonases evolved by fluorescence-activated sorting of single cells in emulsion compartments.

Single bacterial cells, each expressing a different library variant, were compartmentalized in aqueous droplets of water-in-oil (w/o) emulsions, thus maintaining a linkage between a plasmid-borne gene, the encoded enzyme variant, and the fluorescent product this enzyme may generate. Conversion into a double, water-in-oil-in-water (w/o/w) emulsion enabled the sorting of these compartments by FACS, as well as the isolation of living bacteria cells and their enzyme-coding genes. We demonstrate the directed evolution of new enzyme variants by screening >10(7) serum paraoxonase (PON1) mutants, to yield 100-fold improvements in thiolactonase activity. In vitro compartmentalization (IVC) of single cells, each carrying >10(4) enzyme molecules, in a volume of <10 femtoliter (fl), enabled detection and selection despite the fast, spontaneous hydrolysis of the substrate, the very low initial thiolactonase activity of PON1, and the use of difusable fluorescent products.

Aryldialkylphosphatase↗
PubMed1997

Efficiency of single-cell polymerase chain reaction from stained histologic slides and integrity of DNA in archival tissue.

Molecular analysis of isolated single cells is a powerful tool for studying heterogeneity within a population of cells and for clarifying issues of cell origin and clonality. Here, we investigate the applicability of molecular techniques at a single-cell level by using routinely processed archival tissue. An ultraviolet laser in conjunction with a computer-controlled micromanipulator and a microscope were used for the contamination-free isolation of single tumor cells from stained sections of diffuse-type gastric cancer. A total of 1,328 single cells and 654 clusters of 10-30 cells each, taken from specimens of 14 patients, were analyzed for parts of the E-cadherin gene by the polymerase chain reaction (PCR). With increasing length in base pairs (bp) of the amplified fragments, the efficiency of single-cell PCR as measured by the rate of detectable amplification products declined from approximately 25% (156, 213, and 228 bp) to 14% (246 bp) and 11% (264 and 296 bp). For groups of 10-30 cells, a similar effect was seen at a higher level at 33% (246 bp), 31% (264 bp), and 26% (296 bp), respectively. To our knowledge, this is the first report that has studied the outcome of single-cell PCR on a large systematic scale. The average degree of DNA disintegration in paraffin-embedded, stained tissues was estimated to be approximately 100 bp when the aforementioned data were used in a mathematical model. This study provides evidence that in order to obtain reasonable sensitivity with single-cell PCR, short fragments, preferably < 200 bp long, should be used. Furthermore, whenever applicable, pooling of cells of interest may be another favorable option.

DNA↗
PubMed1995

Improved micro-perfusion chamber for multiple and rapid solution exchange in adherent single cells.

A new method for fast perfusion of single adherent cells during whole-cell and single-channel patch-clamp measurements is described. The main advantages over previous methods are: (1) the solution surrounding a single cell in a Petri dish (or cover slip) can be exchanged without contamination of the entire cell population, and (2) only small quantities of test solution (50 microliters) are required. The method consists of inserting a patch pipette into a micro-perfusion chamber (MPC) where whole-cell and single-channel currents can be studied. A small volume (2-3 microliters) surrounding adherent single cells in a Petri dish is separated by enclosing it in a ring of Silicone rubber which is then pressed to the bottom of the dish. A drug-containing test solution (50 microliters) is supplied to a funnel at the chamber inflow. The MPC volume isolated from the main solution in the Petri dish is rapidly changed when suction is applied to the chamber outflow. The speed of solution exchange in the MPC (16 +/- 5 ms, n = 12) was estimated by observing changes in the tip potential of 1-M omega patch pipettes during rapid chamber perfusion with solutions of different ionic composition. The seal of the MPC with the surface of a Petri dish was approximated by measuring the electrical resistance between the MPC interior and a reference electrode placed in the Petri dish outside the MPC (range 300-500 M omega). Additionally, a radioactive calcium channel ligand [3H]isradipine was added to the MPC and the appearance of radioactivity in the Petri dish (outside the MPC) subsequently measured.(ABSTRACT TRUNCATED AT 250 WORDS)

Amplifiers, Electronic↗
PubMed2025

A single-cell transcriptomic atlas of the pigtail macaque placenta in late gestation.

The placenta is a complex organ with multiple immune and non-immune cell types that promote fetal tolerance and facilitate the transfer of nutrients and oxygen. The nonhuman primate (NHP) is a key experimental model for studying human pregnancy complications, in part due to similarities in placental structure, which makes it essential to understand how single-cell populations compare across the human and NHP maternal-fetal interface. We constructed a single-cell RNA-Seq (scRNA-Seq) atlas of the placenta from the pigtail macaque ( Macaca nemestrina ) in the third trimester, comprising three different tissues at the maternal-fetal interface: the chorionic villi (placental disc), chorioamniotic membranes, and the maternal decidua. Each tissue was separately dissociated into single cells and processed through the 10X Genomics and Seurat pipeline, followed by aggregation, unsupervised clustering, and cluster annotation. Next, we determined the maternal-fetal origins of cell populations and analyzed single-cell RNA trajectory, Gene Ontology enrichment, and cell-cell communication. Single-cell populations in the pigtail macaque were strikingly similar in their identity and frequency to those found in the human placenta, including cells from trophoblast, stromal cell, immune, and macrophage lineages. An advantage of our approach was the deep sequencing of three tissues at the maternal-fetal interface, which yielded a rich diversity of common and rare single-cell populations. The third-trimester pigtail macaque single-cell atlas enables the identification of cellular subclusters analogous to those in humans and provides a powerful resource for understanding experimental perturbations on the NHP placenta.

Journal Article↗
PubMed2004

Comparison of genotoxicity of textile dyestuffs in Salmonella mutagenicity assay, in vitro micronucleus assay, and single cell gel/comet assay.

The mutagenicity of textile dyes is an important consideration for the assurance of consumer protection and work safety. The mutagenicity testing of textile dyestuffs is crucial for accurately predicting health risks for consumers and workers exposed to dyes. Unfortunately, these data are often lacking. We studied the genotoxic activity of ten selected commercial textile dyestuffs, which are made up of mixtures of azo dyes and azo metal complex dyes as well as two anthraquinone dyestuffs. We used the Salmonella mutagenicity assay and cultured human keratinocytes (HaCaT cell line). In the S. typhimurium strain TA98, with and without S9, eight often dyestuffs investigated, and in strain TA 100, with and without S9, six often dyes caused frameshift mutations and base-pair substitutions in the dose range of 1-5000 microg/plate in a dose-related manner. All dyes, including those negative in the Salmonella mutagenicity assay, induced clastogenic effects in the in vitro micronucleus (MN) test in HaCaT cells as direct-acting mutagens in the concentration range of 5-150 microg/mL and with maximum MN frequencies between 1.1 and 7.2%, compared to negative controls that showed 0.2-0.4% MN cells. In the single cell gel/comet assay, all ten dyestuffs investigated caused DNA damage in HaCaT keratinocytes. The alkaline (pH >13) version used is capable of detecting DNA single strand breaks, alkali-labile sites, and DNA-DNA/DNA-protein cross-linking. Under the conditions of these screening tests, the textile dyes investigated are direct-acting genotoxic substances. The HaCaT cells testing protocol proposed has been shown to be an appropriate test system for evaluating mutagenicity of textile dyes on a base level.

Coloring Agents↗
PubMed2026

Pesci: fast and user-friendly software to compare single-cell gene expression across species.

SUMMARY: Recent technological advances have propelled comparative functional genomics into the single-cell era, spurring a rapid development of methods to analyse these complex datasets. However, comparing single-cell gene expression across species to quantify expression similarity and ultimately identify homologous cell types remains an open problem. The ICC algorithm (Iterative Correlation of Coexpression) has been recently proposed as an attractive approach to tackle this challenge, but, to date, no software implementation is available. Here, we introduce Pesci (Pretty Easy Single-cell Comparisons using ICC), an efficient and user-friendly implementation of the ICC algorithm applied to pairwise comparisons of single-cell gene expression atlases across species. AVAILABILITY: Pesci is implemented in Python 3 (&#x2265;3.7). It is available for download on Linux, macOS and Windows via pip, conda and GitHub at https://github.com/eparey/pesci. The source code is permanently archived on Zenodo (https://doi.org/10.5281/zenodo.21477543).

Software↗
PubMed2004

Flow cytometric analysis of protein content in Taxus protoplasts and single cells as compared to aggregated suspension cultures.

Plant suspension cultures are highly aggregated, preventing the direct application of flow cytometry for the study of population dynamics. The utility of single cells to accurately represent aggregated suspension cultures was tested through the analysis of total protein content. Specifically, protein content of two Taxus cuspidata suspension culture lines was studied using the Bradford assay for aggregated suspension cultures, and flow cytometry with fluorescein isothiocyanate staining for protoplasts and single cells. Taxus protein levels were measured at 75-160 mg per gram dry weight via the Bradford assay. Aggregated suspension cultures, protoplasts, and single cells predicted the same trend of protein content over the culture period (21 days). Normalized protein content of isolated single cells was statistically equivalent to aggregated suspensions for both cell lines. However, normalized protein content of isolated protoplasts showed significant differences from aggregated suspensions for one of the two cell lines. Elicitation with methyl jasmonate (MJ) is commonly utilized to increase paclitaxel accumulation in suspension cultures, and therefore the effect of MJ elicitation on protein content in aggregated suspensions, isolated single cells and protoplasts was assessed. Aggregated suspension cultures, protoplasts, and single cells did not show any change in total protein content following elicitation with MJ at 200 microM on day 7. This study illustrates the usefulness of flow cytometry for obtaining culture population information and the value of using intact single cells for the study of plant metabolism.

Cell Aggregation↗
PubMed2003

A simple DNA extraction and rapid specific identification technique for single cells and early embryos of two breeds of Bos taurus.

In the process of nuclear transfer (NT), different cytoplasm from a donor cell and a recipient oocyte are mixed. However, it is unclear what effect the donor cytoplasm has upon the dedifferentiation of donor nuclei in enucleated ooplasm and upon subsequent production of live cloned offspring. Mitochondria are component parts of cytoplasm so the detection of mitochondrial DNA is helpful to reveal changes of donor cytoplasm in the NT reconstructed embryos. In this study, the experiments were designed to develop efficient DNA extraction techniques and specific primer pairs for mitochondrial DNA of Holstein and Chinese Yellow breeds in order to identify the changes of donor cytoplasm in early stage embryos. Firstly, by adding Triton X-100 and Taq DNA polymerase reaction buffer to the DNA extraction mixture, DNA was rapidly isolated from single diploid cells, single oocytes, early stage embryos and from single hairs. Secondly, two specific primer pairs for the two breeds were designed to detect the cytoplasmic DNA in a different amount of single cells and in early stage embryos. The results show that two specific fragments were successfully amplified from single somatic cells, single oocytes, parthenogenetic embryos and from NT reconstructed embryos. As a result, the techniques provide a powerful tool for studying the developmental mechanism in NT reconstructed embryos.

Animals↗