Volume: 10 Issue: 19 —BIO-PROTOCOL

Biophysics

Assembly and Imaging Set up of PIE-Scope Authors: Sergey Gorelick, David A. Dierickx, Genevieve Buckley, James C. Whisstock and Alex de Marco, date: 10/05/2020, view: 1213, Q&A: 0

Cryo-Electron Tomography (cryo-ET) is a method that enables resolving the structure of macromolecular complexes directly in the cellular environment. However, sample preparation for in situ Cryo-ET is labour-intensive and can require both cryo-lamella preparation through cryo-Focused Ion Beam (FIB) milling and correlative light microscopy ...

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Cancer Biology

A Novel and Robust Single-cell Trapping Method on Digital Microfluidics Authors: Jiao Zhai, Haoran Li, Ada Hang-Heng Wong, Cheng Dong, Shuhong Yi, Yanwei Jia, Pui-In Mak, Chuxia Deng and Rui Martins, date: 10/05/2020, view: 908, Q&A: 0

Due to cell heterogeneity, the differences among individual cells are averaged out in bulk analysis methods, especially in the analysis of primary tumor biopsy samples from patients. To deeply understand the cell-to-cell variation in a primary tumor, single-cell culture and analysis with limited amount of cells are in high demand. Microfluidics ...

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Cell Biology

Affinity Purification of GO-Matryoshka Biosensors from E. coli for Quantitative Ratiometric Fluorescence Analyses Authors: Mayuri Sadoine, Vanessa Castro-Rodríguez, Tobias Poloczek, Helene Javot, Erdem Sunal, Michael M. Wudick and Wolf B. Frommer, date: 10/05/2020, view: 1382, Q&A: 0

Genetically encoded biosensors are powerful tools for quantitative visualization of ions and metabolites in vivo. Design and optimization of such biosensors typically require analyses of large numbers of variants. Sensor properties determined in vitro such as substrate specificity, affinity, response range, dynamic range, and ...

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Double Labeling of PDGFR-β and α-SMA in Swine Models of Acute Kidney Injury to Detect Pericyte-to-Myofibroblast Transdifferentation as Early Marker of Fibrosis Authors: Alessandra Stasi, Rossana Franzin, Chiara Divella, Loreto Gesualdo, Giovanni Stallone and Giuseppe Castellano, date: 10/05/2020, view: 948, Q&A: 0

Growing evidences suggest that peritubular capillaries pericytes are the main source of scar-forming myofibroblasts during chronic kidney disease (CKD), as well as early phases of acute kidney injury (AKI). In a swine model of sepsis and I/R (Ischemia Reperfusion) injury-induced AKI we demonstrated that renal pericytes are able to ...

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Developmental Biology

Fluorescence Measurement and Calibration of Intracellular pH in Starfish Oocytes Authors: Enako Hosoda and Kazuyoshi Chiba, date: 10/05/2020, view: 1119, Q&A: 0

Oocyte maturation is a process wherein an oocyte arrested at prophase of meiosis I resumes meiosis to become a fertilizable egg. In starfish ovaries, a hormone released from follicle cells activates the oocytes, resulting in an increase in their intracellular pH (pH_i), which is required for spindle assembly. Herein, we describe a ...

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Microbiology

Radioactive Assay of in vitro Glutamylation Activity of the Legionella pneumophila Effector Protein SidJ Authors: Alan G. Sulpizio, Jung-Ho Shin, Marena E. Minelli and Yuxin Mao, date: 10/05/2020, view: 1159, Q&A: 0

The Legionella effector protein SidJ has recently been identified to perform polyglutamylation on another Legionella effector, SdeA, ablating SdeA’s activity. SidJ is a kinase-like protein that requires the small eukaryotic protein calmodulin to perform glutamylation. Glutamylation is a relatively uncommon type of ...

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TetR Regulated in vivo Repression Technology to Identify Conditional Gene Silencing in Genetically Engineerable Bacteria Using Vibrio cholerae Murine Infections as Model System Authors: Franz G. Zingl, Fabian Mitterer, Himadri B. Thapa and Stefan Schild, date: 10/05/2020, view: 859, Q&A: 0

Investigation of bacterial gene regulation upon environmental changes is still a challenging task. For example, Vibrio cholerae, a pathogen of the human gastrointestinal tract, faces diverse transient conditions in different compartments upon oral ingestion. Genetic reporter systems have been demonstrated to be extremely powerful tools to ...

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Analysis of Gram-negative Bacteria Peptidoglycan by Ultra-performance Liquid Chromatography Authors: Laura Alvarez, Baptiste Cordier, Sven van Teeffelen and Felipe Cava, date: 10/05/2020, view: 1416, Q&A: 0

Bacteria are surrounded by a protective peptidoglycan cell wall. Provided that this structure and the enzymes involved are the preferred target for our most successful antibiotics, determining its structural and chemical complexity is of the highest interest. Traditionally, high-performance liquid chromatography (HPLC) analyses have been ...

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Simple Time-lapse Imaging for Quantifying the Hydrostatic Production of Oxygenic Photogranules Authors: Esmee D. Joosten, Jérôme Hamelin and Kim Milferstedt, date: 10/05/2020, view: 1061, Q&A: 0

Oxygenic photogranules (OPGs) are dense, three-dimensional aggregates containing a syntrophic, light-driven microbial community. Their temporal and spatial development interests microbial ecologists working at the bioprocess engineering interface, as this knowledge can be used to optimize biotechnological applications, such as wastewater treatment ...

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Molecular Biology

Using RNA Sequencing and Spike-in RNAs to Measure Intracellular Abundance of lncRNAs and mRNAs Authors: Megan D. Schertzer, McKenzie M. Murvin and J. Mauro Calabrese, date: 10/05/2020, view: 1412, Q&A: 0

Long noncoding RNAs (lncRNAs) play essential roles in normal physiology and in disease but their mechanisms of action can be challenging to identify. For mechanistic studies, it is often useful to know a lncRNA’s intracellular abundance, i.e., approximately how many molecules of the lncRNA are present in a typical cell of a cell-type of ...

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Neuroscience

Preparing Viable Hippocampal Slices from Adult Mice for the Study of Sharp Wave-ripples Authors: Linhua Liu, Xiaojing Zhou and Jian-young Wu, date: 10/05/2020, view: 1280, Q&A: 0

We describe a protocol for preparing acute brain slices which can produce robust hippocampal sharp wave-ripples (SWRs) in vitro. The protocol is optimized for its simplicity and reliability for the preparation of solutions, slicing, and recovery incubation. Most slices in almost every mouse prepared though the protocol expressed vigorous ...

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An Operant Conditioning Model Combined with a Chemogenetic Approach to Study the Neurobiology of Food Addiction in Mice Authors: Elena Martín-García, Laura Domingo-Rodriguez and Rafael Maldonado, date: 10/05/2020, view: 1305, Q&A: 0

The study of food addiction comprises 3 hallmarks that include the persistence to response without an outcome, the strong motivation for palatable food, and the loss of inhibitory control over food intake that leads to compulsive behavior in addicted individuals. The complex multifactorial nature of this disorder and the unknown neurobiological ...

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Plant Science

A Protocol for Flavonols, Kaempferol and Quercetin, Staining in Plant Root Tips Author: Nguyen Hoai Nguyen, date: 10/05/2020, view: 1388, Q&A: 0

Flavonols are a subclass of flavonoids of the group of plant secondary metabolites. In planta, flavonols play various functions such as antioxidant and natural regulator of auxin polar transport. Many lines of evidence have shown that flavonols also contribute to human health in anti-oxidation, anti-inflammation, and even prevention some ...

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Multitarget Immunohistochemistry for Confocal and Super-resolution Imaging of Plant Cell Wall Polysaccharides Authors: Kalina T. Haas, Methieu Rivière, Raymond Wightman and Alexis Peaucelle, date: 10/05/2020, view: 2251, Q&A: 0

The plant cell wall (PCW) is a pecto-cellulosic extracellular matrix that envelopes the plant cell. By integrating extra-and intra-cellular cues, PCW mediates a plethora of essential physiological functions. Notably, it permits controlled and oriented tissue growth by tuning its local mechano-chemical properties. To refine our knowledge of these ...

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Stem Cell

Fluidigm Based Single-cell Gene Expression Library Preparation from Patient-derived Small Intestinal Organoids Authors: Kohei Suzuki and Ryuichi Okamoto, date: 10/05/2020, view: 985, Q&A: 0

In this protocol, we describe our methods to isolate crypts from patients' biopsy samples and to culture human intestinal stem cells as it’s called “organoid.” Beyond that, we describe how to dissociate organoids cells into single cells for single-cell analysis as a further application. This protocol should provide investigators sufficient tools ...

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