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At least 73 records · Page 4Linked to original sources
PubMed1984

Mechanism of cell-mediated cytotoxicity at the single cell level. VI. Direct assessment of the cytotoxic potential of human peripheral blood non-lytic effector-target cell conjugates.

Single cell cytotoxicity assays reveal that a large percentage of lymphocytes are unable to kill attached targets in a 4- to 18-hr assay. Additional signals (in the form of lectin or anti-target antibody) delivered to target-bound lymphocytes enable these previously non-lytic lymphocytes to kill attached target cells. This finding was obtained by using a modification of the single cell assay, in which lectin or target cell antibody is incorporated into agarose with preformed lymphocyte-target conjugates. Human peripheral blood lymphocytes (PBL) or Percoll density gradient-enriched large granular lymphocytes (LGL) were used as effector cells in natural killer (NK), antibody-dependent cellular cytotoxicity (LDCC) assay systems. The targets used were NK-sensitive K562 and Molt-4 and NK-insensitive Raji. Several findings were made in the modified single cell assay, namely a) the frequency of cytotoxic NK or ADCC effector cells was not augmented, suggesting that the initial trigger was sufficient for lytic expression in these instances. Furthermore, these results showed that the NK-sensitive targets used do not bind nonspecifically to the LDCC effector cells. K562 coated with Con A, however, serve as LDCC targets. b) The frequency of two target conjugate lysis by NK/K effectors was not augmented by Con A. These results suggest that Con A does not potentiate the killing of multiple targets bound to a single cytotoxic lymphocyte. c) Although conjugates formed between LGL or PBL and NK-insensitive Raji are non-lethal, significant lysis was observed when these conjugates were suspended in Con A or antibody agarose. These results demonstrate that Raji bind to cytotoxic NK, K, and LDCC effector cells, but are lysed only when the appropriate trigger is provided. d) The cytotoxic potential of non-lytic conjugates appears to lie within the low density Percoll fraction, although the high density lymphocytes are able to nonlethally bind to targets. Altogether the results demonstrate that target recognition and/or binding by the effector cells is a distinct event from the trigger or lytic process. The implications of these findings are discussed.

Animals↗
PubMed2005

Identification of P2Y receptor subtypes in human muller glial cells by physiology, single cell RT-PCR, and immunohistochemistry.

PURPOSE: Retinal Müller glial cells are known to express metabotropic P2Y receptors. The present study was conducted to identify certain subtypes of P2Y receptors in human Müller cells. METHODS: The patch-clamp technique was used to measure increases of Ca(2+)-dependent K+ currents mediated by the activation of P2Y receptors in freshly isolated human Müller cells. Several P2 agonists were used. Subsequently, the cells were harvested into the patch pipette and a single cell RT-PCR was performed. Moreover, retinal tissue from organ donors was used for immunohistochemistry. RESULTS: The electrophysiological data were consistent with the expression of P2Y1, P2Y2, P2Y4, and P2Y6 receptor subtypes. RT-PCR revealed that mRNA for all these subtypes was present in Müller cells. However, the incidence of P2Y2 receptor mRNA was significantly lower than that of the other subtypes. Immunoreactivity for all four subtypes was found in retinal tissue, partly colocalized with immunoreactivity for vimentin. CONCLUSIONS: The presented data obtained by different techniques revealed that human Müller cells express P2Y1, P2Y2, P2Y4, and P2Y6 receptors. The specific roles of these receptor subtypes in retinal physiology and/or pathophysiology remain to be investigated in future studies.

Fluorescent Antibody Technique, Indirect↗
PubMed2006

Sorting of cells of the same size, shape, and cell cycle stage for a single cell level assay without staining.

BACKGROUND: Single-cell level studies are being used increasingly to measure cell properties not directly observable in a cell population. High-performance data acquisition systems for such studies have, by necessity, developed in synchrony. However, improvements in sample purification techniques are also required to reveal new phenomena. Here we assessed a cell sorter as a sample-pretreatment tool for a single-cell level assay. A cell sorter is routinely used for selecting one type of cells from a heterogeneous mixture of cells using specific fluorescence labels. In this case, we wanted to select cells of exactly the same size, shape, and cell-cycle stage from a population, without using a specific fluorescence label. RESULTS: We used four light scatter parameters: the peak height and area of the forward scatter (FSheight and FSarea) and side scatter (SSheight and SSarea). The rat pheochromocytoma PC12 cell line, a neuronal cell line, was used for all experiments. The living cells concentrated in the high FSarea and middle SSheight/SSarea fractions. Single cells without cell clumps were concentrated in the low SS and middle FS fractions, and in the higher FSheight/FSarea and SSheight/SSarea fractions. The cell populations from these viable, single-cell-rich fractions were divided into twelve subfractions based on their FSarea-SSarea profiles, for more detailed analysis. We found that SSarea was proportional to the cell volume and the FSarea correlated with cell roundness and elongation, as well as with the level of DNA in the cell. To test the method and to characterize the basic properties of the isolated single cells, sorted cells were cultured in separate wells. The cells in all subfractions survived, proliferated and differentiated normally, suggesting that there was no serious damage. The smallest, roundest, and smoothest cells had the highest viability. There was no correlation between proliferation and differentiation. NGF increases cell viability but decreases the proliferative ability of the PC12 cells. CONCLUSION: We demonstrated a pretreatment method to collect well-characterized, viable, single cells without using fluorescent labels and without significant damage to the cells. This method is quantitative, rapid, single-step, and yields cells of high purity, making it applicable for a variety of single-cell level analyses.

Adrenal Gland Neoplasms↗
PubMed2026

scFANCL: Dual contrastive learning with false-negative correction at cell level for single-cell RNA-seq clustering.

BACKGROUND: Single-cell RNA sequencing (scRNA-seq) enables cellular characterization at single-cell resolution. However, its high dimensionality, sparsity, and noise make clustering challenging. Approaches utilizing contrastive learning and data augmentation have been introduced to improve representation quality for scRNA-seq clustering. In particular, dual contrastive frameworks combining instance- and cluster-level objectives can capture both cell-cell similarities and inter-cluster variations. However, existing dual contrastive frameworks focus primarily on discrete cluster boundaries, neglecting the biological continuity inherent in scRNA-seq data. METHODS: We propose scFANCL, a dual contrastive framework designed to capture biological continuity in scRNA data. Rather than treating all non-augmented samples as negatives, scFANCL applies a cosine-similarity-based threshold to exclude cells of the same type from the negative pool, preserving continuous transcriptional relationships among them while maintaining inter-cluster separation. RESULTS: Extensive experiments across seven publicly available scRNA-seq datasets demonstrated that scFANCL achieves competitive clustering performance compared with existing baseline methods, consistently yielding high ARI and NMI scores across datasets of varying size and complexity. Ablation studies further confirmed the contribution of the false negative filtering component, showing measurable improvements over variants without filtering. Downstream analyses further suggest that the learned embeddings may reflect biologically meaningful transcriptional transitions, including continuous differentiation trajectories within related cell types. The source code is available at https://github.com/mjuailab/scFANCL . CONCLUSIONS: scFANCL addresses a key limitation of conventional contrastive learning by applying a cosine-similarity-based threshold to exclude cells of the same type from the negative pool, thereby preserving biological continuity within cell types while maintaining inter-cluster separation. Evaluations across seven benchmark scRNA-seq datasets demonstrate competitive clustering performance, with learned embeddings capturing biologically meaningful transcriptional structure and characteristics of rare cell populations.

Clustering Algorithms↗
PubMed2026

Multiomic study of cutaneous T-cell lymphoma reveals single-cell clonal evolution in progression and therapy resistance.

Cutaneous T-cell lymphoma (CTCL) remains a challenging disease due to its significant heterogeneity, therapy resistance, and relentless progression. Multiomics technologies offer the potential to provide uniquely precise views of disease progression and response to therapy. Here, we present a comprehensive multiomics view of CTCL clonal evolution, incorporating exome, whole-genome, epigenome, bulk, single-cell T-cell receptor, and single-cell RNA sequencing of 99 clinically annotated serial skin, peripheral blood, and lymph node samples from 34 patients with CTCL. We leveraged this extensive data set to define the molecular underpinnings of CTCL progression in individual patients at single-cell resolution with the goal of identifying clinically useful biomarkers and therapeutic targets. Our studies identified recurrent progression-associated clonal genomic alterations; we highlight mutation of CCR4, phosphoinositide 3-kinase inhibitor signaling, and programmed cell death protein 1 (PD-1) checkpoint pathways as evasion tactics deployed by malignant T cells. We identified a gain-of-function mutation in STAT3 (D661Y) and demonstrated, using cleavage under targets and release using nuclease (CUT&RUN) and RNA sequencing, that it enhances binding to and transcription of genes in Rho GTPase pathways. With our previous work implicating this pathway in histone deacetylase inhibitor-resistant CTCL, these data provide further support for a previously unrecognized role for Rho GTPase pathway dysregulation in CTCL progression. Recurrent progression-associated mutations were common in the epigenetic modifier EZH2, suggesting that EZH2 inhibition may benefit patients with CTCL. Our findings support an approach in which genomic analysis is widely used for improved disease monitoring, biomarker-informed clinical trial design, and genome-guided therapeutic decision-making. Moreover, these molecular changes present new opportunities for therapeutic targeting in this challenging and incurable cancer.

Multiomics↗
PubMed1983

Electrofusion of myeloma cells on the single cell level. Fusion under sterile conditions without proteolytic enzyme treatment.

A technique is presented which allows electrofusion of single cells under sterile conditions. The electrofusion chamber is placed in a Petri dish. Before a droplet of the fusion medium is pipetted between the electrodes, the chamber is completely covered with vaseline, which prevents the fusion medium evaporating. Additionally, the fusion chamber is treated with solutions containing poly(L)-lysine and pronase which results in a decreased movement of the cells on the glass between the electrodes and which allows electrofusion without any proteolytic pretreatment.

Animals↗
PubMed2000

Coexpression patterns of mGLuR mRNAs in rat retinal ganglion cells: a single-cell RT-PCR study.

PURPOSE: Eight different subunits of metabotropic glutamate receptors (mGluRs) are known to date. mGluRs have been linked to an extensive list of neuromodulatory effects, depending on which intracellular or membrane-bound effector system is activated. Activation of mGluRs can influence neuronal activity and can result in changes of intracellular Ca2+ homeostasis-that is, changes in factors that are known to be crucial for cellular differentiation and cell death. Because mGluRs are known in modulating both intracellular and intercellular activities, this study was designed to determine which types of mGluRs are coexpressed in a neuron and whether distinct coexpression patterns can be found that reflect the different physiological requirements of a neuron at different stages of development and to learn whether neuronal injury results in adaptive changes of mGluR expression. METHODS: Juvenile and adult rat retinal ganglion cells (RGCs) and adult RGCs after axotomy were analyzed for their gene expression pattern of mGluRs by single-cell reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: Adult RGCs predominantly expressed one or two different mGluR mRNAs, whereas juvenile RGCs coexpressed two and more. mGluR3, -5, and -7 mRNAs were found more frequently in juvenile than in adult RGCs. mGluR6 was detected in juvenile RGCs in low abundance but never in adult RGCs. However, mGluR6 was expressed in adult RGCs after axotomy. mGluR1 and -7 were also found more frequently in axotomized RGCs than in the adult control group. CONCLUSIONS: All types of mGluR mRNAs are expressed in RGCs. This is in contrast with previous in situ hybridization and immunohistochemical studies in which expression of mGluR3, -5, and -6 was not reported. The expression of some mGluR mRNAs seems to be developmental, although no distinct copatterns were found.

Aging↗
PubMed2000

Radiation doses to the cell nucleus in single cells and cells in micrometastases in targeted therapy with (131)I labeled ligands or antibodies.

PURPOSE: The aim of this study was to theoretically investigate how the radiation dose to cell nuclei depends on the subcellular position of (131)I. The influence of the size of the cells and crossfire irradiation in clusters of cells was also studied. METHODS AND MATERIAL: Using data describing the dose rate around a point source of (131)I, we calculated the dose distributions inside and around cell models of different sizes. The assumed positions of (131)I were on the cellular or nuclear membrane, in the cytoplasm, in the nucleus, or spread in the whole cell. The mean doses to the nucleus of the targeted cell and to the nuclei of its neighbors were calculated using the dose distributions. RESULTS: The dose distributions inside a single targeted cell showed very different distribution profiles depending on the subcellular position of the (131)I. Targeting the nucleus instead of the cellular membrane could increase the dose to the nucleus 10-fold. Crossfire irradiation can be the major contributor to the nuclear dose in clusters of more than six cells. CONCLUSIONS: Dosimetry without microscopic considerations is inadequate for targeted radionuclide therapy of disseminated or clustering tumor cells exposed to (131)I. Therapeutic doses could be achieved, even in single cells, when (131)I was positioned near, or inside the cell nucleus, or when the clusters were large enough.

Cell Count↗
PubMed2003

Mechanism of activation of human peripheral blood NK cells at the single cell level by Echinacea water soluble extracts: recruitment of lymphocyte-target conjugates and killer cells and activation of programming for lysis.

Echinacea purpurea, a plant originally used by native Americans to treat respiratory infections, has also been shown to exert immunomodulatory activities both in vivo and in vitro. However, the mechanism underlying Echinacea-induced immunomodulation remains largely unknown. This study examined in vitro the effects of soluble extracts of E. purpurea on natural killer (NK) cells present in human peripheral blood mononuclear cells (PBMC). Flow cytometric methods were used to examine activation, cytotoxicity, NK-target binding, and killer cell frequency. Treatment of PBMC with Echinacea overnight resulted in the activation of CD69 expression and increase in mean fluorescence intensity in both the CD16+ and CD16+CD56+ NK subsets. However, the frequency of CD16+ cells was decreased as well as the mean fluorescence intensity was down-regulated. NK cytotoxicity was augmented 100% at the concentration of 0.1 microg/ml of Echinacea in a short time (4-h) assay. Examination at the single cell level revealed augmentation of the frequency of CD56+ NK-target conjugates and a plateau was reached after 30-60 min of incubation. Likewise, the frequency of CD56+ killer cells in the conjugates was also significantly increased by Echinacea. There was recruitment of non-conjugated CD56+ cells into CD16+ NK-target conjugates and activation of the NK-target non-killer conjugates into killer cells. These findings demonstrate that Echinacea extracts are potent activators of NK cytotoxicity. Echinacea augments the frequency of NK target conjugates and activates the programming for lysis of NK cells.

Antigens, CD↗
PubMed2000

Clonal evolution in a primary cutaneous follicle center B cell lymphoma revealed by single cell analysis in sequential biopsies.

B cell neoplasias descending from germinal center cells harbor the hallmark of intraclonal diversity resulting from ongoing mutation in the variable parts of their immunoglobulin-encoding genes. To characterize a primary cutaneous follicle center B cell lymphoma in more detail, we analyzed the respective VH and VL genes in single cells mobilized from four sequential biopsies, three taken from the skin and one obtained after internal dissemination from a retrobulbar infiltrate. The lymphoma cells were found to contain V5-51/D6-12/JH5b (heavy chain) and A27/Jkappa2 (light chain) gene rearrangements detected on both the genomic and the transcriptional level. To provide an accurate mutation analysis, the specific VH gene counterpart (V5-51UK) was cloned from the patient's germline. Analyzing 226 single cells, we found: (i) complete nucleotide identity when VH and VL genes of lymphoma cells from one particular biopsy were compared among each other; (ii) intraclonal diversity due to ongoing mutation comparing the sequences obtained from sequential biopsies; (iii) both VH and VL genes to be highly mutated. Deducing from the sequence data, we propose a scenario of the clonal evolution of the B cell tumor in this patient. From the molecular-biological point of view, this primary cutaneous follicle center B cell lymphoma shows the features of a germinal center cell lymphoma. To draw this conclusion from single cell PCR data, however, a sample of sequential biopsies had to be analyzed.

B-Lymphocytes↗
PubMed2006

Dielectrophoresis tweezers for single cell manipulation.

Positioning single cells is of utmost importance in areas of biomedical research as diverse as in vitro fertilization, cell-cell interaction, cell adhesion, embryology, microbiology, stem cell research, and single cell transfection. Here we describe dielectrophoretic tweezers, a sharp glass tip with electrodes on either side, capable of trapping single cells with electric fields. Mounted on a micromanipulator, dielectrophoresis tweezers can position a single cell in three dimensions, holding the cell against fluid flow of hundreds of microns per second with more than 10 pN of force. We model the electric field produced by the tweezers and the field produced by coaxial microelectrodes. We show that cells are trapped without harm while they divide in the trap. In addition, dielectrophoretic tweezers offer the possibility for trapping, electroporating, and microinjecting a single cell with one probe.

Cell Culture Techniques↗
PubMed1998

Antigen-stimulated IL-4, IL-13 and IFN-gamma production by human T cells at a single-cell level.

To understand the intricate balance and the coordinate expression of the Th1 and Th2 cytokines following a natural mode of T cell triggering, antigen-stimulated IL-4, IL-13 and IFN-gamma production was studied in primary peripheral blood mononuclear cell cultures at a single-cell level. Cells from filariasis patients who respond to parasite antigen by producing not only IFN-gamma but also IL-4 and IL-13 were stimulated with Brugia malayi adult worm antigen and analyzed for co-expression of cytokines by intracellular staining. IL-4 and IL-13 were frequently co-expressed (54% of IL-4+ cells stained for IL-13 and 29% of IL-13+ cells expressed IL-4 at all time points), whereas IFN-gamma expression was totally segregated from both IL-4 and IL-13. These data indicate that in human peripheral T cells the co-expression of the dominant Th1 and Th2 cytokines within a single cell is a rare event and that IL-13 is clearly more frequently associated with a Th2 than a Th1 type response in primary T cell cultures.

Animals↗
PubMed2000

Molecular single-cell analysis of the clonal relationship of small Epstein-Barr virus-infected cells and Epstein-Barr virus-harboring Hodgkin and Reed/Sternberg cells in Hodgkin disease.

Epstein-Barr virus (EBV) can be detected in the tumor cells of approximately 40% of cases of classical Hodgkin disease (cHD). Clonality studies suggest that infection of the neoplastic Hodgkin and Reed/Sternberg (HRS) cells occurs before tumor clone expansion. In EBV-positive cases, variable numbers of EBER-positive small B cells are sometimes also observed that immunohistologically differ from the neoplastic cells by lack of CD30 and latent membrane protein 1 expression. To analyze the clonal relationship between these EBV(+) cells and the HRS cells, single EBV-infected CD30(-) B cells, as well as HRS cells from 3 cases of EBV-positive cHD were micromanipulated, their immunoglobulin gene rearrangements amplified and then compared with each other. In 2 cases, all small EBV-infected cells were clonally unrelated to the HRS cells. In a third case, 2 of 29 small CD30(-) cells were found to carry HRS cell-specific rearrangements. Thus, small CD30(-) EBV-infected B cells in cHD belong to the HRS tumor clone rarely, if at all. In all cases, small clones unrelated to the HRS cell clones were identified among the small EBV(+) CD30(-) cells. The vast majority of small EBV(+) CD30(-) B cells was found to carry somatically mutated V region genes, indicating that in lymph nodes of patients with HD, like in the peripheral blood of healthy individuals, EBV persists in memory B cells.

Adult↗
PubMed1987

Circadian rhythmicity within single cells of Paramecium bursaria.

Cell populations of Paramecium bursaria show mating reactivity in the light period, but not in the dark period, when exposed to a light-dark cycle (LD 12:12). After they are transferred to constant-light (LL) conditions (1,000 lux), they continue to show a circadian rhythm of mating reactivity. The rhythm gradually dampens in LL so that mating reactivity in populations becomes arrhythmic in LL within 2 weeks. We wanted to know whether the arrhythmicity of this population was due to the absence of circadian rhythmicity within each individual cell, or merely due to asynchrony of a population of individually rhythmic cells. Therefore, single cells were isolated randomly from an arrhythmic population that had been in LL for a long time. Then the mating reactivity of these single cells was individually tested every 3 hr for 2 days. Each single cell showed a circadian mating rhythm in LL. This shows that the disappearance of the mating rhythm in cell populations under LL is not caused by disappearance of circadian rhythmicity within individual cells, but is due to desynchronization among cells in a population. When an arrhythmic population in LL is darkened for 9 hr, the mating reactivity rhythm of the cell population reappears. This occurs by resynchronization of the rhythms among individual cells, as can be shown by exposing single cells to pulses of 9 hr of darkness. This dark treatment causes phase shifts of single-cell rhythms, and a phase response curve is obtained for this stimulus. This phase-shifting behavior explains the efficacy of 9-hr dark pulses in restoring the population's rhythm.

Animals↗
PubMed1995

Analysis of the heavy chain repertoire of human peripheral B cells using single-cell polymerase chain reaction.

The VH gene repertoire of human peripheral B cells was analyzed using PCR analysis of individual blood B cells. Because genomic DNA of single B cells was analyzed, data from both productive and nonproductive VDJ rearrangements were obtained. Nine out of 75 B cells contained both functional and nonfunctional rearrangement products, whereas 62/75 had a single productive VDJ rearrangement. The distribution of VH families was ordered in accordance with the germline complexity, although a bias toward VH3 and some of its members was found. This bias was noted in both the productively and nonproductively rearranged repertoires, indicating that it resulted from molecular and not selective processes. Evidence for negative selection of certain VH3 and VH4 family members was noted in that they were found less often as productive than nonproductive VDJ rearrangements. In addition, evidence for positive selection based on CDR3 was obtained, in that JH6 and DXP'1 were found at a higher frequency in the productively compared with the nonproductively rearranged repertoire. The CDR3 segments were markedly heterogenous, with a much greater degree of variability noted in the nonproductive VDJ rearrangements. Finally, a much greater frequency of mutations was noted in the nonproductively rearranged VH genes within individual B cells. These results have begun to delineate the human peripheral B cell repertoire and identify processes that shape it both positively and negatively.

B-Lymphocytes↗
PubMed1989

Extracellular ATP mobilizes intracellular Ca2+ in T51B rat liver epithelial cells: a study involving single cell measurements.

T51B rat liver epithelial cells were stimulated with extracellular ATP. Changes in cytoplasmic free Ca2+ concentration [( Ca2+]i) were measured by fura-2 both in a large population of cells on coverslips in a cuvette and in single cells in a microscopic system. Extracellular ATP evoked a prompt increase in [Ca2+]i in both the presence and absence of extracellular Ca2+, although the effect was less pronounced in the latter case. These findings indicate that at least part of the [Ca2+]i increase is due to mobilization of intracellularly bound calcium. Stimulation with ATP did not mobilize the total pool of intracellular releasable Ca2+, as evidenced from experiments where subsequent addition of ionomycin evoked a pronounced increase in [Ca2+]i in the absence of extracellular Ca2+. The effect of ATP was maintained at room temperature but was markedly impaired in the absence of continuous stirring of the buffer solution. In the absence of stirring, ATP had to be increased to the millimolar range in order to evoke a pronounced effect. Single cell measurements revealed a heterogenous Ca2+ response to ATP, with some cells failing to respond with a detectable increase in [Ca2+]i. The actual increase in [Ca2+]i was not uniform throughout the cytoplasm, but seemed to start in one part of the cell. Even if part of the [Ca2+]i increase might be accounted for by ATP promoting the hydrolysis of phosphatidylinositol 4,5-bisphosphate and thereby a generation of InsP3 and diacylglycerol, there was no initiation of DNA synthesis under the present conditions. Hence, extracellular growth factors exert either a quantitative difference in second messenger production or additional stimulatory effects by activating intracellular signal pathways beyond these represented by [Ca2+]i and protein kinase C.

Adenosine Triphosphate↗
PubMed2026

scATAnno: Automated Cell Type Annotation for Single-cell ATAC-seq Data.

Recent advances in single-cell epigenomic techniques have increased the demand for single-cell assay for transposase-accessible chromatin using sequencing (scATAC-seq) analysis. One key analytical task is to determine cell type identity based on epigenetic data. Here, we introduce scATAnno, a Python package designed to automatically annotate scATAC-seq data using large-scale scATAC-seq reference atlases. This workflow generates reference atlases from publicly available datasets, enabling accurate cell type annotation by integrating query data with reference atlases without the use of single-cell RNA sequencing (scRNA-seq) data. To enhance annotation accuracy, we incorporated k-nearest neighbors (KNN)-based and weighted distance-based uncertainty scores to effectively detect cell populations within the query data that are distinct from all cell types in the reference data. We compared and benchmarked scATAnno against five other published cell annotation approaches, demonstrating its superior performance across multiple datasets and metrics. We further showcased the utility of scATAnno across multiple datasets, including peripheral blood mononuclear cells (PBMCs), triple-negative breast cancer (TNBC), and basal cell carcinoma (BCC), and demonstrated that scATAnno accurately annotates cell types across diverse biological conditions. Overall, scATAnno is a useful tool for scATAC-seq reference atlas construction and cell type annotation and can facilitate the interpretation of new scATAC-seq datasets in complex biological systems. scATAnno is publicly available at https://scatanno-main.readthedocs.io/.

Single-Cell Analysis↗
PubMed1997

Ca2+-triggered peptide secretion in single cells imaged with green fluorescent protein and evanescent-wave microscopy.

Green fluorescent protein fused to human chromogranin B or neuropeptide Y was expressed in PC12 cells and caused bright, punctate fluorescence. The fluorescent points colocalized with the endogenous secretory granule marker dopamine beta-hydroxylase. Stimulation of live PC12 cells with elevated [K+], or of permeabilized PC12 cells with Ca2+, led to Ca2+-dependent loss of fluorescence from neurites. Ca2+ stimulated secretion of both fusion proteins equally well. In living cells, single fluorescent granules were imaged by evanescent-wave fluorescence microscopy. Granules were seen to migrate; to stop, as if trapped by plasmalemmal docking sites; and then to disappear abruptly, as if through exocytosis. Evidently, GFP fused to secreted peptides is a fluorescent marker for dense-core secretory granules and may be used for time-resolved microscopy of single granules.

Animals↗