Volume: 9 Issue: 21 —BIO-PROTOCOL

Cancer Biology

Generation of CoilR Probe Peptides for VIPER-labeling of Cellular Proteins Authors: Julia K. Doh, Savannah J. Tobin and Kimberly E. Beatty, date: 11/05/2019, view: 589, Q&A: 0

Versatile Interacting Peptide (VIP) tags are a new class of genetically-encoded tag designed for imaging cellular proteins by fluorescence and electron microscopy. In 2018, we reported the VIPER tag (Doh et al., 2018), which contains two elements: a genetically-encoded peptide tag (i.e., CoilE) and a probe peptide (i.e., ...

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Implementing VIPER for Imaging Cellular Proteins by Fluorescence Microscopy Authors: Julia K. Doh, Caroline A. Enns and Kimberly E. Beatty, date: 11/05/2019, view: 544, Q&A: 0

Genetically-encoded tags are useful tools for multicolor and multi-scale cellular imaging. Versatile Interacting Peptide (VIP) tags, such as VIPER, are new genetically-encoded tags that can be used in various imaging applications. VIP tags consist of a coiled-coil heterodimer, with one peptide serving as the genetic tag and the other (“probe ...

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Imaging VIPER-labeled Cellular Proteins by Correlative Light and Electron Microscopy Authors: Julia K. Doh, Young Hwan Chang, Caroline A. Enns, Claudia S. Lόpez and Kimberly E. Beatty, date: 11/05/2019, view: 592, Q&A: 0

Advances in fluorescence microscopy (FM), electron microscopy (EM), and correlative light and EM (CLEM) offer unprecedented opportunities for studying diverse proteins and nanostructures involved in fundamental cell biology. It is now possible to visualize and quantify the spatial organization of cellular proteins and other macromolecules by FM, ...

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F-actin Bundle Sedimentation Assay Authors: Shan-Shan Lin, Mei-Chun Chuang and Ya-Wen Liu, date: 11/05/2019, view: 575, Q&A: 0

Understanding the molecular mechanism governing the higher-order regulation of actin dynamics requires chemically-defined and quantitative assays. Recently, the membrane remodeling large GTPase, dynamin, has been identified as a new actin cross-linking molecule. Dynamin regulates actin cytoskeleton through binding to, self-assembling around, and ...

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Cell Biology

High Throughput Traction Force Microscopy for Multicellular Islands on Combinatorial Microarrays Authors: Ian C. Berg and Gregory H. Underhill, date: 11/05/2019, view: 766, Q&A: 0

The composition and mechanical properties of the cellular microenvironment along with the resulting distribution of cellular devolved forces can affect cellular function and behavior. Traction Force Microscopy (TFM) provides a method to measure the forces applied to a surface by adherent cells. Numerous TFM systems have been described in ...

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Developmental Biology

Isolation and Culture of Murine Hepatic Stellate Cells Authors: Rucha V. Modak and Dietmar M. Zaiss, date: 11/05/2019, view: 1037, Q&A: 0

Hepatic stellate cells (HSCs), alternatively known as liver pericytes, can differentiate into myofibroblasts and secrete extra-cellular matrix components, thereby promoting wound healing and fibrosis. Studying HSCs can provide insights into the pathological mechanisms governing these processes. HSC isolation methods typically comprise of enzymatic ...

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Immunology

Amplex Red Assay for Measuring Hydrogen Peroxide Production from Caenorhabditis elegans Authors: Ozgur Karakuzu, Melissa R. Cruz, Yi Liu and Danielle A. Garsin, date: 11/05/2019, view: 849, Q&A: 0

Reagents such as Amplex^® Red have been developed for detecting hydrogen peroxide (H₂O₂) and are used to measure the release of H₂O₂ from biological samples such as mammalian leukocytes undergoing the oxidative burst. Caenorhabditis elegans is commonly used as a model host in the study ...

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Adoptive Transfer of Basophils Enriched from Mouse Spleen Authors: Adrian M. Piliponsky and Stephen J. Galli, date: 11/05/2019, view: 520, Q&A: 0

CD49b is a member of the integrin family, expressed on basophils, natural killer (NK) cells and a subset CD4⁺ T cells in the spleen. This protocol describes the adoptive transfer of basophil-enriched CD49b⁺ cells obtained from mouse spleens by magnetic enrichment. This protocol can be used to assess the contribution of ...

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Microbiology

In vitro Screening of Antileishmanial Activity of Natural Product Compounds: Determination of IC₅₀, CC₅₀ and SI Values Authors: Olga S. Koutsoni, Kalliopi Karampetsou and Eleni Dotsika, date: 11/05/2019, view: 428, Q&A: 0

Neglected tropical diseases gain the scientific interest of numerous research programs in an attempt to achieve their effective control or elimination. In this attempt, more cutting-edge public health policies and research are needed for the discovery of new, safer and effective drugs originated from natural products. Here, we describe protocols ...

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Model of Chemotherapy-associated Mucositis and Oral Opportunistic Infections Authors: Takanori Sobue, Martinna Bertolini, Angela Thompson and Anna Dongari-Bagtzoglou, date: 11/05/2019, view: 410, Q&A: 0

Oral mucositis is a common complication of cancer chemotherapy treatment. Because of the lack of relevant oral mucositis experimental models, it is not clear how chemotherapeutic agents injure the oral mucosa and if commensal microorganisms accelerate tissue damage. We developed an organotypic oral mucosa model that mimics cellular responses ...

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A Mismatch-tolerant RT-LAMP Method for Molecular Diagnosis of Highly Variable Viruses Authors: Yingxue Li, Yi Zhou, Yingying Ma, Rong Xu, Xia Jin and Chiyu Zhang, date: 11/05/2019, view: 422, Q&A: 0

Loop-mediated isothermal amplification (LAMP) has been widely used in the detection of pathogens. However, there are usually numerous variants in one viral pathogen and primers employed in LAMP can hardly match all these variants. The mismatches between the primers and the viral genomes, especially those at the 3′-end of the primers, hinder LAMP ...

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Purification and HPLC Analysis of Cell Wall Muropeptides from Caulobacter crescentus Authors: Gabriele Stankeviciute and Eric A. Klein, date: 11/05/2019, view: 498, Q&A: 0

The peptidoglycan sacculus, or cell wall, is what defines bacterial cell shape. Cell wall composition can be best characterized at the molecular level by digesting the peptidoglycan murein polymer into its muropeptide subunits and quantifying the abundance of muropeptides using high-pressure liquid chromatography. Certain features of the cell wall ...

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Molecular Biology

Using Imaging Flow Cytometry to Characterize Extracellular Vesicles Isolated from Cell Culture Media, Plasma or Urine Authors: John R. Woollard, Amrutesh Puranik, Kyra L. Jordan and Lilach O. Lerman, date: 11/05/2019, view: 634, Q&A: 0

The ability to non-invasively detect specific damage to the kidney has been limited. Identification of extracellular vesicles released by cells, especially when under duress, might allow for monitoring and identification of specific cell types within the kidney that are stressed. We have adapted a previously published traditional flow cytometry ...

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Neuroscience

Isolation and Imaging of His- and RFP-tagged Amyloid-like Proteins from Caenorhabditis elegans by TEM and SIM Authors: Amberley D. Stephens, Meng Lu and Gabriele S. Kaminski Schierle, date: 11/05/2019, view: 569, Q&A: 0

In our recently published paper, we highlight that during normal aging of C. elegans age-dependent aggregates of proteins form and lead to functional decline of tissues. The protocol described here details the isolation of two proteins from C. elegans in their aggregated amyloid-like form, casein kinase I isoform alpha (KIN-19) ...

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Stem Cell

Differentiation of Mouse Embryonic Stem Cells to Neuronal Cells Using Hanging Droplets and Retinoic Acid Authors: Jeroen Witteveldt and Sara Macias, date: 11/05/2019, view: 586, Q&A: 0

Controlled differentiation of embryonic stem cells is an essential tool in stem cell research. In this protocol, we describe a simple differentiation protocol involving the induction of embryoid body formation in mouse embryonic stem cells (mESC) using hanging droplets, followed by differentiation into a neuronal lineage.

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Past Issues in 2019

Volume: 9 Issue: 21

Cancer Biology

Cell Biology

Developmental Biology

Immunology

Microbiology

Molecular Biology

Neuroscience

Stem Cell