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| Open AccessThe CRISPR-Cas13a Gemini System for noncontiguous target RNA activation
CRISPR-Cas13a based methods currently use contiguous target RNA activation, which only enables single-target detection or editing. Here the authors propose a noncontiguous target RNA activation approach which can provide rapid, simultaneous and sensitive detection of two RNAs in a single readout, as well as parallel dual transgene knockdown.
- Hongrui Zhao
- , Yan Sheng
- & Jiaming Hu
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Article
| Open AccessDeciphering the mechanism of glutaredoxin-catalyzed roGFP2 redox sensing reveals a ternary complex with glutathione for protein disulfide reduction
Fusion proteins between roGFP2 and glutaredoxins are used for intracellular redox measurements. Here, the authors determined all rate constants of the reaction cycle for roGFP2 measurements and identified an alternative glutaredoxin mechanism.
- Fabian Geissel
- , Lukas Lang
- & Marcel Deponte
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Article
| Open AccessRapid deep learning-assisted predictive diagnostics for point-of-care testing
A key aim in the development of diagnostic assays is improving diagnostic speed while maintaining sensitivity. Here the authors report an approach for the rapid and accurate analysis of lateral flow tests, which integrates time-series deep learning and AI verification, achieving a diagnostic time of 1-2 minutes.
- Seungmin Lee
- , Jeong Soo Park
- & Jeong Hoon Lee
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Article
| Open AccessBiporous silica nanostructure-induced nanovortex in microfluidics for nucleic acid enrichment, isolation, and PCR-free detection
Efficient enrichment and isolation of pathogens are crucial for accurate and sensitive disease identification. Here, the authors present a chip equipped with biporous nanofilms that induces a nanovetex in a microfluidic channel for nucleic acid enrichment, isolation, and detection.
- Eunyoung Jeon
- , Bonhan Koo
- & Joonseok Lee
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Article
| Open AccessTracing back primed resistance in cancer via sister cells
Transcriptional cell states can drive treatment resistance in cancer. Here, the authors develop ReSisTrace to predict cell states that are primed to resist ovarian cancer treatment and validate their findings using small molecule inhibitors.
- Jun Dai
- , Shuyu Zheng
- & Anna Vähärautio
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Article
| Open AccessMammalian cell growth characterisation by a non-invasive plate reader assay
Automated and non-invasive mammalian cell analysis is currently lagging behind due to a lack of methods suitable for a variety of cell lines and applications. Here the authors develop a high throughput non-invasive method for tracking suspension and adhesion mammalian cell growth based on plate reader measures to characterize engineered cell lines.
- Alice Grob
- , Chiara Enrico Bena
- & Francesca Ceroni
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Article
| Open AccessANCA: artificial nucleic acid circuit with argonaute protein for one-step isothermal detection of antibiotic-resistant bacteria
Antibiotic-resistant bacteria pose a growing threat to global health. Here, the authors present an artificial nucleic acid circuit with argonaute protein (ANCA) for one-step, amplification-free, and isothermal detection of carbapenemase-producing Klebsiella pneumoniae.
- Hyowon Jang
- , Jayeon Song
- & Taejoon Kang
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Article
| Open AccessA semiconductor 96-microplate platform for electrical-imaging based high-throughput phenotypic screening
Cell-based phenotypic assays link in vitro discovery to disease pathology. Here, the authors report a semiconductor-based microplate platform to perform high-throughput, high-dimensional “electrical imaging” for label-free assessment of live cell morphology and function.
- Shalaka Chitale
- , Wenxuan Wu
- & Jeffrey Abbott
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Article
| Open AccessAsymmetric CRISPR enabling cascade signal amplification for nucleic acid detection by competitive crRNA
New strategies are being developed to simplify CRISPR-based nucleic acid detection. By investigating the competitive reaction between a full-sized crRNA and split crRNA for CRISPR-Cas12a, the authors develop an asymmetric CRISPR assay for amplification-free, cascade signal amplification detection of nucleic acids.
- Jeong Moon
- & Changchun Liu
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Article
| Open AccessImaging cellular forces with photonic crystals
Current techniques for visualizing cell generated forces suffer from throughput limitations. Here, Gu et al. introduced photonic crystal cellular force microscopy, inspired by chameleons, enabling visualization and quantification of vertically directed cell forces, well-suited for drug screening.
- Qiwei Li
- , Zaozao Chen
- & Zhongze Gu
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Article
| Open AccessNULISA: a proteomic liquid biopsy platform with attomolar sensitivity and high multiplexing
Unlocking the blood proteome requires exquisite sensitivity and multiplexing to detect low and high abundance proteins simultaneously. Here the authors describe a 200-plex immunoassay with attomolar sensitivity to detect important low abundance proteins in inflammatory diseases and COVID-19.
- Wei Feng
- , Joanne C. Beer
- & Xiao-Jun Ma
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Article
| Open AccessBarcoded multiple displacement amplification for high coverage sequencing in spatial genomics
Spatial genomics offers insights into cellular interactions within tissues. Here, the authors develop barcoded multiple displacement amplification, achieving high-coverage sequencing to map complex genomic variations within cellular landscapes.
- Jinhyun Kim
- , Sungsik Kim
- & Sunghoon Kwon
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Article
| Open AccessBiosensor Guided Polyketide Synthases Engineering for Optimization of Domain Exchange Boundaries
Engineering polyketide synthases can be challenging due to the absence of efficient high-throughput methods. Here, the authors used a solubility biosensor to identify stable variants from libraries of modified polyketide synthases.
- Elias Englund
- , Matthias Schmidt
- & Jay D. Keasling
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Article
| Open AccessIdentification of scaffold proteins for improved endogenous engineering of extracellular vesicles
Extracellular vesicles are naturally occurring nanoparticles that are gaining ground as delivery modalities for therapeutics. Here, the authors conducted a large-scale screening programme to identify potential scaffold proteins for cargo loading into extracellular vesicles.
- Wenyi Zheng
- , Julia Rädler
- & Samir EL Andaloussi
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Article
| Open AccessA multiplexed bacterial two-hybrid for rapid characterization of protein–protein interactions and iterative protein design
Protein-protein interactions (PPIs) are crucial for biological functions and have applications ranging from drug design to synthetic cell circuits. Here the authors develop an assay and computational methods to identify more orthogonal coiled-coil pairs, critical for biological processes and drug design.
- W. Clifford Boldridge
- , Ajasja Ljubetič
- & Sriram Kosuri
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Article
| Open AccessEnabling programmable dynamic DNA chemistry using small-molecule DNA binders
The binding of small molecules to the double stranded DNA may significantly alter its stability and functionality, which is the basis for many therapeutic and sensing applications. Here, the authors report that DNA binders can be used to program reaction pathways of a dynamic DNA reaction, where DNA strand displacement can be tuned quantitatively according to the affinity, charge, and concentrations of a given DNA binder.
- Junpeng Xu
- , Guan Alex Wang
- & Feng Li
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Article
| Open AccessExtending the dynamic range of biomarker quantification through molecular equalization
The human plasma proteome spans over 10 orders of magnitude, but current quantification methods cover only 3-4 orders. Here, authors propose a tuning approach for proximity-based assays, enabling simultaneous quantification of four analytes across a wide concentration range (low fM to mid-nM).
- Sharon S. Newman
- , Brandon D. Wilson
- & H. Tom Soh
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Article
| Open AccessFully co-factor-free ClearTau platform produces seeding-competent Tau fibrils for reconstructing pathological Tau aggregates
The authors report a method for producing co-factor-free fibrils from all full-length Tau isoforms. The method paves the way for reconstituting pathology resembling Tau fibrils and enables screening of Tau aggregation-modifying compounds for targeted therapies and PET tracers.
- Galina Limorenko
- , Meltem Tatli
- & Hilal A. Lashuel
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Article
| Open AccessA rapid cell-free expression and screening platform for antibody discovery
Antibody discovery is bottlenecked by the individual expression and evaluation of antigen specific hits. Here, the authors build an antibody screening workflow leveraging cell-free protein synthesis that enables expression and evaluation of hundreds of antibody fragments in less than 24 h.
- Andrew C. Hunt
- , Bastian Vögeli
- & Michael C. Jewett
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Article
| Open AccessA highly sensitive strategy for monitoring real-time proliferation of targeted cell types in vivo
Currently, tracking cell proliferation in vivo is hard. Here the authors report a method where the time series of proliferation of targeted cell types can be monitored in vivo in the same individuals, with no need for animal sacrifice: they use this to assess β-cell and hepatocyte proliferation in mice.
- Hiroto Sugawara
- , Junta Imai
- & Hideki Katagiri
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Article
| Open AccessRAD-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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Article
| Open AccessAll-optical closed-loop voltage clamp for precise control of muscles and neurons in live animals
Optogenetic actuation regimes are often static, which allows perturbation, but not true control of neuronal activity. Here, the authors describe an all-optical method for bidirectional steering of membrane potential, in closed loop, in C. elegans muscles and neurons, and rat hippocampal slice culture. The ‘optogenetic voltage clamp’ uses two microbial rhodopsin actuators and the rhodopsin voltage indicator QuasAr.
- Amelie C. F. Bergs
- , Jana F. Liewald
- & Alexander Gottschalk
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| Open AccessGenome-wide tiled detection of circulating Mycobacterium tuberculosis cell-free DNA using Cas13
In this work, the authors developed a multiplexed, minimally invasive, CRISPR-Cas13-based approach to detect Mycobacterium tuberculosis cell-free DNA in the plasma of active pulmonary tuberculosis patients.
- Sri Gowtham Thakku
- , Jackson Lirette
- & Deborah T. Hung
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Article
| Open AccessMultiplexed analysis of EV reveals specific biomarker composition with diagnostic impact
Multiplexed analyses of near single EVs is currently challenging. Here the authors report the method MASEV, multiplexed analysis of EVs, to interrogate thousands of individual EVs during 5 cycles of multi-channel fluorescence staining for 15 EV biomarkers.
- Joshua D. Spitzberg
- , Scott Ferguson
- & Ralph Weissleder
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Article
| Open AccessDifferential sensing with arrays of de novo designed peptide assemblies
Differential sensing aims to mimic senses such as taste and smell through the use of synthetic receptors. Here, the authors show that arrays of de novo designed peptide assemblies can be used as sensor components to distinguish various analytes and complex mixtures.
- William M. Dawson
- , Kathryn L. Shelley
- & Derek N. Woolfson
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Article
| Open AccessTAPE-seq is a cell-based method for predicting genome-wide off-target effects of prime editor
Methods to predict genome-wide off-target activities of prime editors (PEs) are currently lacking. Here the authors report a cell-based assay, TAgmentation of Prime Editor sequencing (TAPE-seq), that provides genome-wide off-target candidates for PEs.
- Jeonghun Kwon
- , Minyoung Kim
- & Jungjoon K. Lee
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Article
| Open AccessA growth selection system for the directed evolution of amine-forming or converting enzymes
Fast screening of enzymes is key for directed evolution of industrial biocatalysts. Here, the authors report a simple, high-throughput, and low-equipment-dependent growth selection system for engineering three enzymes for synthesis of chiral amines.
- Shuke Wu
- , Chao Xiang
- & Uwe T. Bornscheuer
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Article
| Open AccessUltrafast one-minute electronic detection of SARS-CoV-2 infection by 3CLpro enzymatic activity in untreated saliva samples
Methods for the detection of active viral infection are desirable. Here, the authors report an electrochemical approach targeting a SARS-CoV-2 proteolytic enzyme called 3CLpro, as a marker of active infection.
- Ella Borberg
- , Eran Granot
- & Fernando Patolsky
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Article
| Open AccessInvestigating lytic polysaccharide monooxygenase-assisted wood cell wall degradation with microsensors
It is important to understand the enzymatic degradation of wood biomass by lytic polysaccharide monooxygenase, however, disagreements about the co-substrate exist. Here, the authors use piezo-controlled hydrogen peroxide micro-sensors to demonstrate that even low levels of hydrogen peroxide support the enzymatic degradation of wood cell walls.
- Hucheng Chang
- , Neus Gacias Amengual
- & Roland Ludwig
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Article
| Open AccessImproved immunoassay sensitivity and specificity using single-molecule colocalization
A major challenge of enzyme-linked immunosorbent assays is discriminating true signal from non-specific binding. Here the authors present a Single-Molecule Colocalization Assay (SiMCA) which eliminates such effects, enabling reproducible detection of picomolar protein concentrations.
- Amani A. Hariri
- , Sharon S. Newman
- & H. Tom Soh
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Article
| Open AccessSpatiotemporal-resolved protein networks profiling with photoactivation dependent proximity labeling
Methods to identify protein interaction networks often suffer from poor spatiotemporal resolution. Here the authors present a light-activated proximity labeling method where the protein of interest is fused to the photosensitizer protein miniSOG, allowing temporally resolved labeling of interactors.
- Yansheng Zhai
- , Xiaoyan Huang
- & Gang Li
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Article
| Open AccessA CRISPR-based ultrasensitive assay detects attomolar concentrations of SARS-CoV-2 antibodies in clinical samples
CRISPR diagnostics are routinely used for the detecting nucleic acids, but rarely for clinically important proteins. Here, by translating a CRISPR-based DNA test into an ultrasensitive assay for antibodies, the authors achieve antibody detection from serum samples at attomolar concentrations.
- Yanan Tang
- , Turun Song
- & Feng Li
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Article
| Open AccessCombi-seq for multiplexed transcriptome-based profiling of drug combinations using deterministic barcoding in single-cell droplets
Current screens to assess tumour drug resistance require a large amount of material, normally not available from patients. Here the authors report CombiSeq, a scalable microfluidic workflow to screen hundreds of drug combinations in picoliter-size droplets using transcriptome changes as a readout.
- L. Mathur
- , B. Szalai
- & C. A. Merten
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Article
| Open AccessDetection of neutralizing antibodies against multiple SARS-CoV-2 strains in dried blood spots using cell-free PCR
Neutralizing antibodies are critical for conferring immunity against SARS-CoV-2. Here, Dahn et al. report a PCR assay termed SONIA (Split-Oligonucleotide Neighboring Inhibition Assay) for measuring neutralizing antibodies against multiple SARS-CoV-2 strains in fingerprick dried blood spot samples.
- Kenneth Danh
- , Donna Grace Karp
- & Cheng-ting Tsai
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Article
| Open AccessNanoscale organization of two-dimensional multimeric pMHC reagents with DNA origami for CD8+ T cell detection
MHC-peptide multimers are important reagents for detecting antigen specific T cells. Here the authors show that DNA scaffolds can be used to make MHC-peptide multimers and the avidity controlled so that low abundance or T cells with low affinity TCR can be detected using these reagents.
- Yueyang Sun
- , Lu Yan
- & Hao Pei
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Article
| Open AccessHomogeneous surrogate virus neutralization assay to rapidly assess neutralization activity of anti-SARS-CoV-2 antibodies
Neutralisation assays are key to understanding immune responses to SARS-CoV-2 infection or vaccination. Here, the authors report a surrogate virus neutralization assay called Neu-SATiN, which measures neutralization directly from sera, and allows easy adaptation to variant-specific testing.
- Sun Jin Kim
- , Zhong Yao
- & Shawn C. Owen
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| Open AccessOpen-source personal pipetting robots with live-cell incubation and microscopy compatibility
Liquid handling and pipetting tools can automate repetitive tasks but are far from universally used. Here the authors report the Pipetting Helper Imaging Lid (PHIL), an open-source liquid handling robot designed for inexperienced users, that they use for automated pipetting.
- Philip Dettinger
- , Tobias Kull
- & Timm Schroeder
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Article
| Open AccessCRISPR-mediated multiplexed live cell imaging of nonrepetitive genomic loci with one guide RNA per locus
Three-dimensional (3D) structures of the genome are dynamic, heterogeneous and functionally important. Here the authors present a CRISPR-based approach for labeling the genome at multiple nonrepetitive loci in living cells and to image chromatin loops in the presence and absence of cohesin.
- Patricia A. Clow
- , Menghan Du
- & Albert W. Cheng
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Article
| Open AccessProgrammable design of isothermal nucleic acid diagnostic assays through abstraction-based models
Detecting nucleic acids often requires choosing between different amplification mechanisms. Here the authors present a generalisable and programmable isothermal methodology, demonstrated in clinical applications, including for multiplexed detection of short miRNAs.
- Gaolian Xu
- , Julien Reboud
- & Jonathan M. Cooper
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Article
| Open AccessMicro-mechanical blood clot testing using smartphones
Therapy with anticoagulants requires frequent monitoring. Here the authors describe a proof-of-concept study of a simple and affordable blood clot test that uses a smartphone’s vibration motor and camera to track micro-movements in a single drop of blood.
- Justin Chan
- , Kelly Michaelsen
- & Shyamnath Gollakota
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Article
| Open AccessSpatial transcriptomics using combinatorial fluorescence spectral and lifetime encoding, imaging and analysis
Spatial-omics methods with ease-of-use and high multiplexing are in demand. Here the authors report Multi Omic Single-scan Assay with Integrated Combinatorial Analysis (MOSAICA) which uses Spectral and Fluorescence Lifetime Imaging and Microscopy; they apply this to co-detection of mRNA and protein.
- Tam Vu
- , Alexander Vallmitjana
- & Weian Zhao
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Article
| Open AccessProtocell arrays for simultaneous detection of diverse analytes
Simultaneous detection of multiple analytes from a sample is currently difficult. Here the authors present protocell arrays in a customisable platform integrating cell-free expression with a polymer-based aqueous two-phase system; they use this for detection of chemically diverse targets from biofluids.
- Yan Zhang
- , Taisuke Kojima
- & Mark P. Styczynski
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Article
| Open AccessVirus detection via programmable Type III-A CRISPR-Cas systems
CRISPR-Cas based virus detection assays can be both rapid and sensitive. Here the authors use a Type III-A CRISPR-Cas system to detect SARS-CoV-2.
- Sagar Sridhara
- , Hemant N. Goswami
- & Hong Li
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Article
| Open AccessGlycan remodeled erythrocytes facilitate antigenic characterization of recent A/H3N2 influenza viruses
Here, Broszeit et al. show that circulating A/H3N2 viruses have evolved binding specificity to α2,6-sialosides on extended LacNAc moieties and therefore cannot agglutinate erythrocytes. Applying glycan remodeling allows to install functional receptors on erythrocytes and promotes identification of newly circulating variants to facilitate vaccine design.
- Frederik Broszeit
- , Rosanne J. van Beek
- & Geert-Jan Boons
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Article
| Open AccessSuppressing mosquito populations with precision guided sterile males
A. aegypti is the principal vector for arboviruses that impact on human health and wellbeing. Here the authors use precision guided sterile insect technique—pgSIT—to suppress or eliminate mosquito populations in multigeneration cage experiments.
- Ming Li
- , Ting Yang
- & Omar S. Akbari
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Article
| Open AccessA non-enzymatic, isothermal strand displacement and amplification assay for rapid detection of SARS-CoV-2 RNA
The reliance on enzymes in SARS-CoV-2 RNA detection imposes limits on transport and storage conditions. Here the authors use non-enzymatic isothermal amplification to detect RNA with no need for reverse transcription.
- Mohsen Mohammadniaei
- , Ming Zhang
- & Yi Sun
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Article
| Open AccessSCOPE enables type III CRISPR-Cas diagnostics using flexible targeting and stringent CARF ribonuclease activation
Type III CRISPR-Cas systems recognize and cleave target RNAs and produce signalling molecules. Here the authors discover that both processes are governed by a flexible seed region, ultimately resulting in SCOPE, a SARSCoV-2 diagnostic assay with atto-molar sensitivity.
- Jurre A. Steens
- , Yifan Zhu
- & Raymond H. J. Staals
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Article
| Open AccessLive cell tagging tracking and isolation for spatial transcriptomics using photoactivatable cell dyes
Spatial transcriptomics aims to pair omic data with tissue structure. Here the authors report Spatially PhotoActivatable Colour Encoded Cell Address Tags (SPACECAT) to track and isolate live cells by location; this enables spatially informed downstream assays like scRNA-seq and flow cytometry.
- Alex S Genshaft
- , Carly G. K. Ziegler
- & Alex K. Shalek
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Article
| Open AccessMeasuring expression heterogeneity of single-cell cytoskeletal protein complexes
Existing methods for multimeric protein complex quantification in single cells suffer from limited selectivity and sensitivity. Here the authors report Single-cell protein Interaction Fractionation Through Electrophoresis and immunoassay Readout (SIFTER) and use this to probe the effects of cellular stress.
- Julea Vlassakis
- , Louise L. Hansen
- & Amy E. Herr