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Metabolic heterogeneity of tissue-resident macrophages in homeostasis and during helminth infection
Gauging the in vivo metabolism of immune cells at the single-cell level has proven challenging. Here the authors use spectral flow cytometry to investigate metabolic profiles in tissue-resident macrophages from several organs and changes in response to helminth infection.
- Graham A. Heieis
- , Thiago A. Patente
- & Bart Everts
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SEC-seq: association of molecular signatures with antibody secretion in thousands of single human plasma cells
Linking proteins secreted from individual cells with other cellular information is challenging. Here, authors report a high-throughput method which uses hydrogel nanovials loaded with single cells to link the secretion profile of individual cells with their surface markers and transcriptomic data.
- Rene Yu-Hong Cheng
- , Joseph de Rutte
- & Richard G. James
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RAD-TGTs: high-throughput measurement of cellular mechanotype via rupture and delivery of DNA tension probes
Mechanical forces drive critical cellular processes, but methods to study such cellular forces are typically low-throughput. Here the authors present a method using “Rupture And Deliver” Tension Gauge Tethers, where flow cytometry or sequencing can be used to record the mechanical history of thousands of individual cells.
- Matthew R. Pawlak
- , Adam T. Smiley
- & Wendy R. Gordon
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Immune phenotypes that are associated with subsequent COVID-19 severity inferred from post-recovery samples
The association of specific immune cell types with SARS-CoV-2 severity is not fully understood. Here the authors analyse PBMC from individuals recovered from COVID-19 and propose immune cell signatures post recovery that are associated with disease severity.
- Thomas Liechti
- , Yaser Iftikhar
- & Mario Roederer
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Massive image-based single-cell profiling reveals high levels of circulating platelet aggregates in patients with COVID-19
The authors report the landscape of circulating platelet aggregates in COVID-19 obtained by massive single-cell image-based profiling and temporal monitoring to elucidate the underlying process of COVID-19-assocaited microvascular thrombosis.
- Masako Nishikawa
- , Hiroshi Kanno
- & Keisuke Goda
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AutoSpill is a principled framework that simplifies the analysis of multichromatic flow cytometry data
Flow cytometry allows the simultaneous quantification of many markers in and on a cell, but the analysis of such data is complicated. Here, the authors propose AutoSpill, a framework that facilitates the analysis of such data by automating parts of the analysis and requiring fewer controls.
- Carlos P. Roca
- , Oliver T. Burton
- & Adrian Liston
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A molecular sensor to quantify the localization of proteins, DNA and nanoparticles in cells
Determining the trafficking of intracellular material is commonly done by colocalisation analysis using microscopy. Here the authors monitor trafficking of select cargo by measuring the conversion of quenched SNAP-tag substrates by subcellularly-localised SNAP-tag and detection by flow cytometry.
- Laura I. FitzGerald
- , Luigi Aurelio
- & Angus P. R. Johnston
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Virtual-freezing fluorescence imaging flow cytometry
High throughput imaging flow cytometry suffers from trade-offs between throughput, sensitivity and spatial resolution. Here the authors introduce a method to virtually freeze cells in the image acquisition window to enable 1000 times longer signal integration time and improve signal-to-noise ratio.
- Hideharu Mikami
- , Makoto Kawaguchi
- & Keisuke Goda
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Automated optimized parameters for T-distributed stochastic neighbor embedding improve visualization and analysis of large datasets
Visualisation tools that use dimensionality reduction, such as t-SNE, provide poor visualisation on large data sets of millions of observations. Here the authors present opt-SNE, that automatically finds data set-tailored parameters for t-SNE to optimise visualisation and improve analysis.
- Anna C. Belkina
- , Christopher O. Ciccolella
- & Jennifer E. Snyder-Cappione
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Functional analysis of genetic variants in the high-risk breast cancer susceptibility gene PALB2
PALB2 is an established breast cancer risk gene but the pathogenicity of many variants remains uncharacterised. Here, the authors present a cDNA-based system for the functional analysis of PALB2 variants of unknown significance.
- Rick A. C. M. Boonen
- , Amélie Rodrigue
- & Haico van Attikum
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Single cell analysis of kynurenine and System L amino acid transport in T cells
Kynurenine is an immunomodulatory aryl hydrocarbon receptor (AHR) ligand. Here the authors show, using a new flow cytometry based method, that kynurenine needs to be transported across the plasma membrane of activated T cells by the transporter protein SLC7A5 to activate the AHR.
- Linda V. Sinclair
- , Damien Neyens
- & Doreen A. Cantrell
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Visual analysis of mass cytometry data by hierarchical stochastic neighbour embedding reveals rare cell types
Single cell profiling yields high dimensional data of very large numbers of cells, posing challenges of visualization and analysis. Here the authors introduce a method for analysis of mass cytometry data that can handle very large datasets and allows their intuitive and hierarchical exploration.
- Vincent van Unen
- , Thomas Höllt
- & Boudewijn P. F. Lelieveldt
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Flow cytometry combined with viSNE for the analysis of microbial biofilms and detection of microplastics
The interpretation of high-dimensional flow cytometry data is difficult and time consuming. Here, Sgier et al.present an improved method that combines stain-free flow cytometry with viSNE data visualization for the analysis of microbial biofilms.
- Linn Sgier
- , Remo Freimann
- & Alexandra Kroll
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Optical painting and fluorescence activated sorting of single adherent cells labelled with photoswitchable Pdots
Isolation of individual cells from mixed populations is desirable for many biomedical applications. Here the authors use photoswitchable Pdots to allow 'optical painting', where cells of interest are marked based on their visual characteristics, and can then be isolated by fluorescence activated cell sorting.
- Chun-Ting Kuo
- , Alison M. Thompson
- & Daniel T. Chiu
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Label-free cell cycle analysis for high-throughput imaging flow cytometry
Imaging flow cytometry enables high-throughput acquisition of fluorescence, brightfield and darkfield images of biological cells. Here, Blasi et al.demonstrate that applying machine learning algorithms on brightfield and darkfield images can detect cellular phenotypes without the need for fluorescent stains, enabling label-free assays.
- Thomas Blasi
- , Holger Hennig
- & Paul Rees
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Ultrasensitive quantification of TAP-dependent antigen compartmentalization in scarce primary immune cell subsets
Antigenic peptides are loaded into major histocompatibility complex class I (MHC I) molecules after entering the ER via the TAP transporter. Here, Fischbach et al. develop a flow cytometry assay to monitor TAP-dependent peptide translocation that is sensitive enough to use on scarce primary cell subsets.
- Hanna Fischbach
- , Marius Döring
- & Robert Tampé
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Sorting of small infectious virus particles by flow virometry reveals distinct infectivity profiles
Individual viral particles can display heterogeneity in their composition of lipids, proteins or nucleic acids. Here, the authors apply advanced flow cytometry techniques to sort Junin virus particles and show that particle size and envelope glycoprotein concentration correlate with infectivity.
- Raphaël Gaudin
- & Natasha S Barteneva
Single drop cytometry onboard the International Space Station