Cytological techniques

  • Article
    | Open Access

    Elucidating specific effects of protein kinase Akt isoforms remains challenging. Here, the authors establish an Akt isoform-dependent cellular model system and use it, together with X-ray crystallography and structure-based ligand design, to develop isoform-selective covalent-allosteric Akt inhibitors

    • Lena Quambusch
    • , Laura Depta
    •  & Daniel Rauh
  • Article
    | Open Access

    During Plasmodium intra-erythrocytic developmental, parasites compromise the structural integrity of host red-blood cells. Here, Clark et al. develop a flow cytometric osmotic stability assay to show that P. vivax infection destabilizes host reticulocytes, which are less stable than P. falciparum-infected normocytes.

    • Martha A. Clark
    • , Usheer Kanjee
    •  & Manoj T. Duraisingh
  • Article
    | Open Access

    It is challenging to map complex processes in brain tissue. Here the authors report a toolkit enabling large-scale multiplexed IHC and automated cell classification whereby they use a conventional epifluorescence microscope and deep neural networks to phenotype all major cell classes of the brain.

    • Dragan Maric
    • , Jahandar Jahanipour
    •  & Badrinath Roysam
  • Article
    | Open Access

    While cell shape is crucial for function and development of organisms, versatile frameworks for cell shape quantification, comparison, and classification remain underdeveloped. Here, the authors use a network-based framework for Arabidopsis leaf epidermal cell shape characterization and classification.

    • Jacqueline Nowak
    • , Ryan Christopher Eng
    •  & Zoran Nikoloski
  • Article
    | Open Access

    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
  • Article
    | Open Access

    The ability to culture live pancreatic tissue slices for long periods of time would enable longitudinal studies ex vivo. Here the authors culture human and mouse pancreatic slices in a perfluorocarbon-based culture system and show stable endocrine and exocrine function for up to ten days in culture.

    • Mirza Muhammad Fahd Qadir
    • , Silvia Álvarez-Cubela
    •  & Juan Domínguez-Bendala
  • Article
    | Open Access

    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
  • Article
    | Open Access

    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
  • Article
    | Open Access

    Meiotic crossovers (COs) are essential for proper chromosome segregation and generating novel combinations of alleles. Here, the authors develop haplotype-specific oligos on maize chromosome 10 for fluorescence in situ hybridization and analyze CO patterns in an intermated recombinant population derived from B73 and Mo17.

    • Lívia do Vale Martins
    • , Fan Yu
    •  & Jiming Jiang
  • Article
    | Open Access

    Autophagic clearance of aggregates, aggrephagy, is essential for cellular homeostasis but tools to induce and monitor it are limited. Here the authors present a fluorescence-based aggrephagy induction assay to study spatiotemporal dynamics and control mechanisms driving protein aggregate clearance.

    • Anne F. J. Janssen
    • , Eugene A. Katrukha
    •  & Lukas C. Kapitein
  • Article
    | Open Access

    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
  • Article
    | Open Access

    Personalised medicine requires cell cultures from defined genetic backgrounds, but providing sufficient numbers of cells is a challenge. Here the authors develop gene cocktails to expand primary cells from a variety of different tissues and species, and show that expanded endothelial and hepatic cells retain properties of the differentiated phenotype.

    • Christoph Lipps
    • , Franziska Klein
    •  & Tobias May
  • Article
    | Open Access

    Induced pluripotent stem cells (iPSCs) have potential for regenerative medicine applications, but are generated with very low efficiency. Here, the authors show highly efficient reprogramming of human primary fibroblasts to iPSCs via the synergistic activity of synthetic modified mRNAs, mature miRNA mimics, and optimized culture methods.

    • Igor Kogut
    • , Sandra M. McCarthy
    •  & Ganna Bilousova
  • Article
    | Open Access

    Little is known about how geometric cues affect cell function and gene expression in 3D settings. Here the authors use microniches of different geometries to control cell volume and shape, and by extension cell phenotype and lineage.

    • Min Bao
    • , Jing Xie
    •  & Wilhelm T. S. Huck
  • Article
    | Open Access

    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
  • Article
    | Open Access

    3D cell culture is more relevant than the two-dimensional format, but methods for parallel analysis and temporal regulation of the microenvironment are limited. Here the authors develop a droplet microfluidics system to perform long-term culture of 3D spheroids, enabling multiscale cytometry of individual cells within the spheroid.

    • Sébastien Sart
    • , Raphaël F.-X. Tomasi
    •  & Charles N. Baroud
  • Article
    | Open Access

    Screening libraries of genetically engineered microbes for secreted products is limited by the available assay throughput. Here the authors combine aptamer-based fluorescent detection with droplet microfluidics to achieve high throughput screening of yeast strains engineered for enhanced tyrosine or streptavidin production.

    • Joseph Abatemarco
    • , Maen F. Sarhan
    •  & Adam R. Abate
  • Article
    | Open Access

    Hepatocytes are highly specialized cells and their fate is determined by their position in the liver as either periportal or perivenous hepatocytes. Here, Pu et al. show through genetic lineage tracing for Mfsd2 that periportal hepatocytes proliferate and reprogram into pericentral hepatocytes during liver regeneration and injury.

    • Wenjuan Pu
    • , Hui Zhang
    •  & Bin Zhou
  • Article
    | Open Access

    The centrosome is a large intracellular structure that serves as the microtubule-organising center, but how it is accurately assembled is not known. Here the authors generate a ‘domain-level’ centrosome interactome and show that Plk4 positions the essential centriole component Asterless by phosphorylating Cep135.

    • Brian J. Galletta
    • , Carey J. Fagerstrom
    •  & Nasser M. Rusan
  • Article
    | Open Access

    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
  • Article
    | Open Access

    Apoptosis often requires mitochondrial outer membrane permeabilization, a process targeted by Bcl-2-binding BH3 mimetics. Here the authors describe and apply 'mito-priming', a method that allows triggering mitochondrial apoptosis in a synchronous manner, facilitating the investigation of mitochondrial apoptosis and its regulation by Bcl-2 proteins.

    • Jonathan Lopez
    • , Margaux Bessou
    •  & Stephen W. G. Tait
  • Article
    | Open Access

    The direct observation of intracellular processes through microscopy can provide key insight into biological pathways. Here the authors demonstrate the combination of microfluidic cell squeezing and fluorescent label delivery to native proteins for high throughput live cell super-resolution imaging.

    • Alina Kollmannsperger
    • , Armon Sharei
    •  & Robert Tampé
  • Article
    | Open Access

    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
  • Article
    | Open Access

    Research on the gut microbiota would benefit from improved methods to study microbial population growth. Here, Myhrvold et al. present a ‘mark and recapture’ method that uses genetically encoded fluorescent particles to measure the growth rates of gut microbes in live animals.

    • Cameron Myhrvold
    • , Jonathan W. Kotula
    •  & Pamela A. Silver
  • Article
    | Open Access

    Multiple extracellular matrix parameters influence cellular behaviour, but it is difficult to dissect their cooperative contributions. Here the authors describe a hydrogel system in which ligand density and substrate stiffness can be tuned orthogonally to study the contribution of combinations of these parameters simultaneously.

    • Andrew D. Rape
    • , Mikhail Zibinsky
    •  & Sanjay Kumar
  • Article
    | Open Access

    The ability to measure signalling responses in single cells following short pulses of stimulus would shed insight into temporal thresholds for cell activation. Here the authors introduce a microfluidic platform that allows downstream phosphorylation cascades to be observed following as little as one second of stimulus exposure.

    • Alphonsus H. C. Ng
    • , M. Dean Chamberlain
    •  & Aaron R. Wheeler
  • Article
    | Open Access

    Despite methodological improvements, it remains extremely difficult to routinely establish cell lines from human primary tumours. Here, the authors develop a culture medium that allows efficient cell line establishment and use this to generate 25 ovarian cancer cell lines.

    • Tan A. Ince
    • , Aurea D. Sousa
    •  & Gordon B. Mills
  • Article |

    Characterizing mammalian gene expression regulation by enhancer elements is complicated by the size and complexity of the genome. Here Vanhille et al.demonstrate CapStarr-Seq, a novel high-throughput method for assessing potential enhancers and deciphering the mechanisms regulating transcription

    • Laurent Vanhille
    • , Aurélien Griffon
    •  & Salvatore Spicuglia
  • Article |

    The ciliary margin of the eye functions as a source of multipotent progenitor cells in certain organisms but whether it plays this role in humans has not been easy to study. Here the authors culture human embryonic stem cells that self-organize into retinal tissue, and show that ciliary margin-like growth zones emerge from the developing human retinal tissue and contain stem cell niches.

    • Atsushi Kuwahara
    • , Chikafumi Ozone
    •  & Yoshiki Sasai
  • Article
    | Open Access

    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
  • Article |

    Tumour microenvironment affects the outcome of pharmacological anticancer treatments. Here, Kenny et al. show that organotypic cultures of ovarian cancer cells can recapitulate metastasis. They identify several new compounds that block cancer invasion and metastasis and improve survival in mouse models.

    • Hilary A. Kenny
    • , Madhu Lal-Nag
    •  & Ernst Lengyel
  • Article |

    The incorporation of foreign objects into cells can be used in various avenues of biological research, although crossing the cell membrane can be challenging. Here, the authors use a diamond nanoneedle array for enhanced delivery of various particles into cells, including neurons.

    • Ying Wang
    • , Yang Yang
    •  & Peng Shi
  • Article
    | Open Access

    Numerous obstacles posed by cellular subcompartments and structures constrain protein transport in the cell. Here, Baum et al.map the intracellular topology from a diffusing protein’s point of view by measuring the diffusive movements of fluorescently labelled reporter proteins in living cells on multiple time and length scales.

    • Michael Baum
    • , Fabian Erdel
    •  & Karsten Rippe
  • Article
    | Open Access

    3D cell culture matrices more closely resemble the natural microenvironments of stem cells than 2D systems. Here, the authors present a 3D cell culture approach to screen for the influence of environmental parameters on self-renewal and differentiation of single mouse embryonic stem cells.

    • A. Ranga
    • , S. Gobaa
    •  & M. P. Lutolf
  • Article |

    Many tissues can be grown as 3D spheroid models in hanging drops of media. Here, Frey et al. develop a microfluidic, interconnected hanging drop network to facilitate inter-drop communication, and demonstrate that pro-drug conversion by liver spheroids can limit the growth of cancer spheroids in adjacent drops.

    • Olivier Frey
    • , Patrick M. Misun
    •  & Andreas Hierlemann
  • Article
    | Open Access

    The three germ layers are formed from the inner cell mass of the mammalian embryo during gastrulation. Here, the authors present a method by which a single mouse embryonic stem cell, derived from inner cell mass, differentiates into the three germ layers in a self-organized manner when cultured in soft fibrin gel.

    • Yeh-Chuin Poh
    • , Junwei Chen
    •  & Ning Wang
  • Article |

    The assessment of nanomaterial toxicity can be hampered by difficulties in determining cell dosage. Here, the authors present a simple method for determination of nanomaterial agglomerate density in liquid media, enabling accurate calculation of dose delivered to cells in an in vitrosystem.

    • Glen DeLoid
    • , Joel M. Cohen
    •  & Philip Demokritou
  • Article |

    Cellular heterogeneity is a feature of diverse disease processes, yet is masked in typical biochemical assays. Sarkar et al.develop a microfluidic device to access the contents of single cells in adherent culture, allowing biochemical measurements to be connected with phenotypic information.

    • Aniruddh Sarkar
    • , Sarah Kolitz
    •  & Jongyoon Han