scholarly journals Single‐Cell Cytokine Assays: Multiplexed, Sequential Secretion Analysis of the Same Single Cells Reveals Distinct Effector Response Dynamics Dependent on the Initial Basal State (Adv. Sci. 9/2019)

2019 ◽  
Vol 6 (9) ◽  
pp. 1970055
Author(s):  
Zhuo Chen ◽  
Yao Lu ◽  
Kerou Zhang ◽  
Yang Xiao ◽  
Jun Lu ◽  
...  
Keyword(s):  
Single Cell ◽  
Single Cells ◽  
Response Dynamics ◽  
Effector Response

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

2019 ◽  
Vol 6 (9) ◽  
pp. 1801361 ◽  
Author(s):  
Zhuo Chen ◽  
Yao Lu ◽  
Kerou Zhang ◽  
Yang Xiao ◽  
Jun Lu ◽  
...  
Keyword(s):  
Single Cells ◽  
Response Dynamics ◽  
Effector Response

scholarly journals Two-level modeling approach to identify the regulatory dynamics capturing drug response heterogeneity in single-cells

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Madalena Chaves ◽  
Luis C. Gomes-Pereira ◽  
Jérémie Roux
Keyword(s):  
Drug Resistance ◽  
Single Cell ◽  
Cell Response ◽  
Drug Response ◽  
Single Cells ◽  
Reaction Model ◽  
Population Heterogeneity ◽  
Cellular Heterogeneity ◽  
Response Dynamics ◽  
Modeling Approach

AbstractSingle-cell multimodal technologies reveal the scales of cellular heterogeneity impairing cancer treatment, yet cell response dynamics remain largely underused to decipher the mechanisms of drug resistance they take part in. As the phenotypic heterogeneity of a clonal cell population informs on the capacity of each single-cell to recapitulate the whole range of observed behaviors, we developed a modeling approach utilizing single-cell response data to identify regulatory reactions driving population heterogeneity in drug response. Dynamic data of hundreds of HeLa cells treated with TNF-related apoptosis-inducing ligand (TRAIL) were used to characterize the fate-determining kinetic parameters of an apoptosis receptor reaction model. Selected reactions sets were augmented to incorporate a mechanism that leads to the separation of the opposing response phenotypes. Using a positive feedback loop motif to identify the reaction set, we show that caspase-8 is able to encapsulate high levels of heterogeneity by introducing a response delay and amplifying the initial differences arising from natural protein expression variability. Our approach enables the identification of fate-determining reactions that drive the population response heterogeneity, providing regulatory targets to curb the cell dynamics of drug resistance.

scholarly journals Multiplexed, sequential secretion analysis of the same single cells reveals distinct effector response dynamics dependent on the initial basal state

2019 ◽  
Author(s):  
Zhuo Chen ◽  
Yao Lu ◽  
Kerou Zhang ◽  
Yang Xiao ◽  
Jun Lu ◽  
...  
Keyword(s):  
Immune Cells ◽  
Time Course ◽  
Single Cells ◽  
Human Macrophage ◽  
Transcriptional Level ◽  
Multiple Time ◽  
Response Dynamics ◽  
Macrophage Response ◽  
Time Points ◽  
Effector Response

AbstractThe 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 we show 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. It was applied to investigating the time course of single human macrophage response to Toll-like receptor 4 (TLR4) ligand lipopolysaccharide and revealed four distinct activation modes for different proteins in single cells. In particular, we observed that secreted factors regulated by transcription factor NFkB (e.g., TNF and CCL2) predominantly show on-off mode over off-on mode. The dynamics of all proteins combined classified the cells into two major activation states, which were found to be dependent on the basal state of each cell. Single-cell RNA-Seq was performed on the same samples at the matched time points and further demonstrated at the transcriptional level the existence of two major activation states, which are enriched for translation vs inflammatory programs, respectively. These results showed a cell-intrinsic heterogeneous response in phenotypically homogeneous cell population. This work demonstrated 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.

scholarly journals Functional single cell proteomic profiling of cells with abnormal DNA damage response dynamics

2021 ◽  
Author(s):  
Pin-Rui Su ◽  
Li You ◽  
Cecile Beerens ◽  
Karel Bezstarosti ◽  
Jeroen Demmers ◽  
...  
Keyword(s):  
Dna Damage ◽  
Single Cell ◽  
Dna Damage Response ◽  
Cell Tracking ◽  
Single Cells ◽  
Proteomic Profiling ◽  
Response Dynamics ◽  
Isolated Cells ◽  
Cellular Dynamics ◽  
Damage Response

Tumor heterogeneity is an important source of cancer therapy resistance. Single cell proteomics has the potential to decipher protein content leading to heterogeneous cellular phenotypes. Single-Cell ProtEomics by Mass Spectrometry (SCoPE-MS) is a recently developed, promising, unbiased proteomic profiling techniques, which allows profiling several tens of single cells for >1000 proteins per cell. However, a method to link single cell proteomes with cellular behaviors is needed to advance this type of profiling technique. Here, we developed a microscopy-based functional single cell proteomic profiling technology, called FUNpro, to link the proteome of individual cells with phenotypes of interest, even if the phenotypes are dynamic or the cells of interest are sparse. FUNpro enables one i) to screen thousands of cells with subcellular resolution and monitor (intra)cellular dynamics using a custom-built microscope, ii) to real-time analyze (intra)cellular dynamics of individual cells using an integrated cell tracking algorithm, iii) to promptly isolate the cells displaying phenotypes of interest, and iv) to single cell proteomically profile the isolated cells. We applied FUNpro to proteomically profile a newly identified small subpopulation of U2OS osteosarcoma cells displaying an abnormal, prolonged DNA damage response (DDR) after ionizing radiation (IR). With this, we identified PDS5A and PGAM5 proteins contributing to the abnormal DDR dynamics and helping the cells survive after IR.

In-Situ Dual Beam (FIBSEM) Techniques for Probe Pad Deposition and Dielectric Integrity Inspection in 0.2 μm Technology DRAM Single Cells

1999 ◽  
Author(s):  
Gunnar Zimmermann ◽  
Richard Chapman
Keyword(s):  
Electron Beam ◽  
Single Cell ◽  
Single Cells ◽  
Ion Beam ◽  
Gate Oxide ◽  
Ion Milling ◽  
Dual Beam ◽  
Sem Imaging ◽  
Innovative Techniques

Abstract Dual beam FIBSEM systems invite the use of innovative techniques to localize IC fails both electrically and physically. For electrical localization, we present a quick and reliable in-situ FIBSEM technique to deposit probe pads with very low parasitic leakage (Ipara < 4E-11A at 3V). The probe pads were Pt, deposited with ion beam assistance, on top of highly insulating SiOx, deposited with electron beam assistance. The buried plate (n-Band), p-well, wordline and bitline of a failing and a good 0.2 μm technology DRAM single cell were contacted. Both cells shared the same wordline for direct comparison of cell characteristics. Through this technique we electrically isolated the fail to a single cell by detecting leakage between the polysilicon wordline gate and the cell diffusion. For physical localization, we present a completely in-situ FIBSEM technique that combines ion milling, XeF2 staining and SEM imaging. With this technique, the electrically isolated fail was found to be a hole in the gate oxide at the bad cell.

scholarly journals Visually precise, low-damage, single-cell spatial manipulation with single-pixel resolution

2021 ◽  
Vol 12 (11) ◽  
pp. 4111-4118
Author(s):  
Qi Zhang ◽  
Yunlong Shao ◽  
Boye Li ◽  
Yuanyuan Wu ◽  
Jingying Dong ◽  
...  
Keyword(s):  
Single Cell ◽  
Single Cells ◽  
Pixel Resolution ◽  
Single Pixel

We achieved the low-damage spatial puncture of single cells at specific visual points with an accuracy of <65 nm.

scholarly journals Single cell transcriptome atlas of mouse mammary epithelial cells across development

2021 ◽  
Vol 23 (1) ◽  
Author(s):  
Bhupinder Pal ◽  
Yunshun Chen ◽  
Michael J. G. Milevskiy ◽  
François Vaillant ◽  
Lexie Prokopuk ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Single Cell ◽  
Developmental Stages ◽  
Mammary Epithelial Cells ◽  
Expression Profiles ◽  
Single Cells ◽  
Chromatin Accessibility ◽  
Mammary Epithelial ◽  
Cell Transcriptome ◽  
Single Cell Transcriptome

Abstract Background Heterogeneity within the mouse mammary epithelium and potential lineage relationships have been recently explored by single-cell RNA profiling. To further understand how cellular diversity changes during mammary ontogeny, we profiled single cells from nine different developmental stages spanning late embryogenesis, early postnatal, prepuberty, adult, mid-pregnancy, late-pregnancy, and post-involution, as well as the transcriptomes of micro-dissected terminal end buds (TEBs) and subtending ducts during puberty. Methods The single cell transcriptomes of 132,599 mammary epithelial cells from 9 different developmental stages were determined on the 10x Genomics Chromium platform, and integrative analyses were performed to compare specific time points. Results The mammary rudiment at E18.5 closely aligned with the basal lineage, while prepubertal epithelial cells exhibited lineage segregation but to a less differentiated state than their adult counterparts. Comparison of micro-dissected TEBs versus ducts showed that luminal cells within TEBs harbored intermediate expression profiles. Ductal basal cells exhibited increased chromatin accessibility of luminal genes compared to their TEB counterparts suggesting that lineage-specific chromatin is established within the subtending ducts during puberty. An integrative analysis of five stages spanning the pregnancy cycle revealed distinct stage-specific profiles and the presence of cycling basal, mixed-lineage, and 'late' alveolar intermediates in pregnancy. Moreover, a number of intermediates were uncovered along the basal-luminal progenitor cell axis, suggesting a continuum of alveolar-restricted progenitor states. Conclusions This extended single cell transcriptome atlas of mouse mammary epithelial cells provides the most complete coverage for mammary epithelial cells during morphogenesis to date. Together with chromatin accessibility analysis of TEB structures, it represents a valuable framework for understanding developmental decisions within the mouse mammary gland.

scholarly journals SUGAR-seq enables simultaneous detection of glycans, epitopes, and the transcriptome in single cells

2021 ◽  
Vol 7 (8) ◽  
pp. eabe3610
Author(s):  
Conor J. Kearney ◽  
Stephin J. Vervoort ◽  
Kelly M. Ramsbottom ◽  
Izabela Todorovski ◽  
Emily J. Lelliott ◽  
...  
Keyword(s):  
Single Cell ◽  
Posttranslational Modification ◽  
Cellular Differentiation ◽  
Single Cells ◽  
Simultaneous Detection ◽  
Surface Protein ◽  
Rna Seq ◽  
Cell Level ◽  
Simultaneous Integration ◽  
Unique Surface

Multimodal single-cell RNA sequencing enables the precise mapping of transcriptional and phenotypic features of cellular differentiation states but does not allow for simultaneous integration of critical posttranslational modification data. Here, we describe SUrface-protein Glycan And RNA-seq (SUGAR-seq), a method that enables detection and analysis of N-linked glycosylation, extracellular epitopes, and the transcriptome at the single-cell level. Integrated SUGAR-seq and glycoproteome analysis identified tumor-infiltrating T cells with unique surface glycan properties that report their epigenetic and functional state.

scholarly journals Nanopore sequencing of single-cell transcriptomes with scCOLOR-seq

2021 ◽  
Author(s):  
Martin Philpott ◽  
Jonathan Watson ◽  
Anjan Thakurta ◽  
Tom Brown ◽  
Tom Brown ◽  
...  
Keyword(s):  
Single Cell ◽  
Error Detection ◽  
Single Cells ◽  
Fusion Transcript ◽  
Building Blocks ◽  
Myeloma Cell ◽  
Nanopore Sequencing ◽  
Long Read ◽  
Unique Molecular Identifier ◽  
Transcript Detection

AbstractHere we describe single-cell corrected long-read sequencing (scCOLOR-seq), which enables error correction of barcode and unique molecular identifier oligonucleotide sequences and permits standalone cDNA nanopore sequencing of single cells. Barcodes and unique molecular identifiers are synthesized using dimeric nucleotide building blocks that allow error detection. We illustrate the use of the method for evaluating barcode assignment accuracy, differential isoform usage in myeloma cell lines, and fusion transcript detection in a sarcoma cell line.

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