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Multiplexed, Sequential Secretion Analysis of the Same Single Cells Reveals Distinct Effector Response Dynamics Dependent on the Initial Basal State

ABSTRACT: The effector response of immune cells is a highly dynamic process. To explore time course of human macrophage response to toll-like receptor 4 (TLR4) ligand, human U937 cell line-derived macrophages were stimulated with lipopolysaccharide (LPS) and single-cell RNA-Sequencing were conducted at 0, 2, 4 and 6 hours after the initial treatment. The data demonstrated the existence of two major activation states at the transcriptional level, which were enriched for translation versus inflammatory programs, respectively. These results show a cell-intrinsic heterogeneous response in a phenotypically homogeneous cell population, providing dynamic information to better understand how immune cells react to pathogenic challenges over time and how they together constitute a population response.

ORGANISM(S): Homo sapiens

PROVIDER: GSE200324 | GEO | 2022/04/09

REPOSITORIES: GEO

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Multiplexed, Sequential Secretion Analysis of the Same Single Cells Reveals Distinct Effector Response Dynamics Dependent on the Initial Basal State.

Project description:The effector response of immune cells dictated by an array of secreted proteins is a highly dynamic process, requiring sequential measurement of all relevant proteins from single cells. Herein, a microchip-based, 10-plexed, sequential secretion assay on the same single cells and at the scale of ?5000 single cells measured simultaneously over 4 time points are shown. It is applied to investigating the time course of single human macrophage response to toll-like receptor 4 (TLR4) ligand lipopolysaccharide (LPS) and reveals four distinct activation modes for different proteins in single cells. Protein secretion dynamics classifies the cells into two major activation states dependent on the basal state of each cell. Single-cell RNA sequencing performed on the same samples at the matched time points further demonstrates the existence of two major activation states at the transcriptional level, which are enriched for translation versus inflammatory programs, respectively. These results show a cell-intrinsic heterogeneous response in a phenotypically homogeneous cell population. This work demonstrates the longitudinal tracking of protein secretion signature in thousands of single cells at multiple time points, providing dynamic information to better understand how individual immune cells react to pathogenic challenges over time and how they together constitute a population response.

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Multiplexed, Sequential Secretion Analysis of the Same Single Cells Reveals Distinct Effector Response Dynamics Dependent on the Initial Basal State

Project description:Multiplexed, Sequential Secretion Analysis of the Same Single Cells Reveals Distinct Effector Response Dynamics Dependent on the Initial Basal State

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SSSMuG: Same Sample Sequential Multi-Glycomics

Project description:The mammalian glycome is structurally complex and diverse, composed of many glycan classes such as N- and O-linked glycans, glycosaminoglycans (GAGs), glycosphingolipids (GSLs) and other distinct glycan features such as polysialic acids (PolySia), sulfation and proteoglycan attachment stubs. Various methods are used to analyse these different components of the glycome, but they require pre-fractionated/partitioned samples to target each glycan class individually. To address this need for a knowledge of the relationship between the different glycan components of a biological system, we have developed a sequential release workflow for analysis of multiple conjugated glycan classes (PolySia, GAGs, GSL glycans, N-glycans O-glycans) from the same tissue lysate, termed SSSMuG: Same Sample Sequential Multi-Glycomics. With this sequential glycan release approach, five glycan classes were characterized (or four glycan classes plus proteomics) from a single sample using enzymatic or chemical release from the same spot immobilized on a polyvinylidene difluoride membrane. The various released glycan classes were then analysed by HPLC and/or MS techniques. Compared to single glycan class release approaches, SSSMuG is able to identify more glycans and more proteins with higher intensity analytical peaks and provides a better comparative normalisation of the different glycan classes of the complex glycome.

2024-02-13 | PXD046012 | Pride

Multiplexed protein profiling by sequential affinity capture.

Project description:Antibody microarrays enable parallelized and miniaturized analysis of clinical samples, and have proven to provide novel insights for the analysis of different proteomes. However, there are concerns that the performance of such direct labeling and single antibody assays are prone to off-target binding due to the sample context. To improve selectivity and sensitivity while maintaining the possibility to conduct multiplexed protein profiling, we developed a multiplexed and semi-automated sequential capture assay. This novel bead-based procedure encompasses a first antigen capture, labeling of captured protein targets on magnetic particles, combinatorial target elution and a read-out by a secondary capture bead array. We demonstrate in a proof-of-concept setting that target detection via two sequential affinity interactions reduced off-target contribution, while lowered background and noise levels, improved correlation to clinical values compared to single binder assays. We also compared sensitivity levels with single binder and classical sandwich assays, explored the possibility for DNA-based signal amplification, and demonstrate the applicability of the dual capture bead-based antibody microarray for biomarker analysis. Hence, the described concept enhances the possibilities for antibody array assays to be utilized for protein profiling in body fluids and beyond.

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Dynamic gene expression response to altered gravity in human myelomonocytic U937 cells

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Multiplexed In-cell Immunoassay for Same-sample Protein Expression Profiling.

Project description:In-cell immunoassays have become a valuable tool for protein expression analysis complementary to established assay formats. However, comprehensive molecular characterization of individual specimens has proven challenging and impractical due to, in part, a singleplex nature of reporter enzymes and technical complexity of alternative assay formats. Herein, we describe a simple and robust methodology for multiplexed protein expression profiling on the same intact specimen, employing a well-characterized enzyme alkaline phosphatase for accurate quantification of all targets of interest, while overcoming fundamental limitations of enzyme-based techniques by implementing the DNA-programmed release mechanism for segregation of sub-sets of target-bound reporters. In essence, this methodology converts same-sample multi-target labeling into a set of isolated singleplex measurements performed in a parallel self-consistent fashion. For a proof-of-principle, multiplexed detection of three model proteins was demonstrated on cultured HeLa cells, and two clinically-relevant markers of dementia, β-amyloid and PHF-tau, were profiled in formalin-fixed paraffin embedded brain tissue sections, uncovering correlated increase in abundance of both markers in the "Alzheimer's disease" cohort. Featuring an analytically powerful yet technically simple and robust methodology, multiplexed in-cell immunoassay is expected to enable insightful same-sample protein profiling studies and become broadly adopted in biomedical research and clinical diagnostics.

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Homo sapiens

Project description:Dynamic gene expression response to altered gravity in human myelomonocytic U937 cells [parabolic]

| PRJNA393965 | ENA

B27 modulation of LPS response

Project description:Objectives How HLA-B27 contributes towards arthritis susceptibility is still unclear, but effects on the response to bacteria unrelated to the classical antigen presenting role of B27 have been suggested. This study investigated whether HLA-B27 modulates the innate response to LPS, a component shared between all Gram –ve bacteria that can trigger reactive arthritis. Methods Pools of U937 transfectants expressing either HLA-B27, HLA-A2, or the expression plasmid alone were differentiated with PMA and stimulated with LPS. Supernatants were analysed for TNF-alpha secretion and the gene expression profiles of unstimulated and LPS stimulated cells were determined by microarray analysis. Changes in gene expression that are indicative of an unfolded protein response were also analysed by quantitative PCR. Results TNF-alpha secretion, a biological marker of the inflammatory response to LPS, was not significantly different between U937-B27 and U937-control. No differences in gene expression between unstimulated U937- B27 and U937-control lines were detected. Both U937-control and U937-B27 exhibited a stereotypic response to LPS. Only 1 gene, OAS2, was differentially expressed by these cell lines, and this was confirmed by quantitative PCR. Analysis of XBP-1 splicing suggested that a small increase in the unfolded protein response is induced following LPS stimulation, but this increase was seen in all transfectants. Conclusions The expression of B27 does not profoundly alter gene expression following LPS stimulation. Therefore, additional signals, such as those provided by cytokines or intracellular infection, may be required to reveal any influence of B27 expression on the inflammatory response. Keywords: antigen response

2006-04-10 | GSE4579 | GEO

Benchmarking of single cell proteomics data analysis workflows

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2024-02-02 | PXD046211 | Pride

Homo sapiens

Project description:Dynamic gene expression response to altered gravity in human myelomonocytic U937 cells [sounding rocket]

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