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Past Issues in 2017

Volume: 7 Issue: 5

Cancer Biology

Ca²⁺ Measurements in Mammalian Cells with Aequorin-based Probes Authors: Anna Tosatto, Rosario Rizzuto and Cristina Mammucari, date: 03/05/2017, view: 2354, Q&A: 0

Aequorin is a Ca²⁺ sensitive photoprotein suitable to measure intracellular Ca²⁺ transients in mammalian cells. Thanks to recombinant cDNAs expression, aequorin can be specifically targeted to various subcellular compartments, thus allowing an accurate measurement of Ca²⁺ uptake and release of different ...

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Liposome Flotation Assays for Phosphoinositide-protein Interaction Authors: Helene Tronchere and Frederic Boal, date: 03/05/2017, view: 4367, Q&A: 0

Phosphoinositides are rare membrane lipids involved in the control of the major cellular functions and signaling pathways. They are able to recruit specific effector proteins to the cytosolic face of plasma membrane and organelles to coordinate a vast variety of signaling and trafficking processes, as well to maintain specific identity of the ...

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Developmental Biology

Protein Synthesis Rate Assessment by Fluorescence Recovery after Photobleaching (FRAP) Authors: Nikos Kourtis and Nektarios Tavernarakis, date: 03/05/2017, view: 2811, Q&A: 0

Currently available biochemical methods cannot be applied to monitor protein synthesis in specific cells or tissues, in live specimens. Here, we describe a non-invasive method for monitoring protein synthesis in single cells or tissues with intrinsically different translation rates, in live Caenorhabditis elegans animals.

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Microbiology

An HPLC-based Method to Quantify Coronatine Production by Bacteria Authors: Shweta Panchal, Zachary S. Breitbach and Maeli Melotto, date: 03/05/2017, view: 2599, Q&A: 0

Coronatine is a polyketide phytotoxin produced by several pathovars of the plant pathogenic bacterium Pseudomonas syringae. It is one of the most important virulence factors determining the success of bacterial pathogenesis in the plant at both epiphytic and endophytic stages of the disease cycle. This protocol describes an optimized ...

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Polyethylene Glycol-mediated Transformation of Drechmeria coniospora Authors: Le D. He and Jonathan J. Ewbank, date: 03/05/2017, view: 2676, Q&A: 0

Drechmeria coniospora is a nematophagous fungus and potential biocontrol agent. It belongs to the Ascomycota. It is related to Hirsutella minnesotensis, another nematophagous fungus but, phylogenetically, it is currently closest to the truffle parasite Tolypocladium ophioglossoides. Together with its natural host, ...

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Next-generation Sequencing of the DNA Virome from Fecal Samples Authors: Cynthia L. Monaco and Douglas S. Kwon, date: 03/05/2017, view: 3209, Q&A: 0

Herein we describe a detailed protocol for DNA virome analysis of low input human stool samples (Monaco et al., 2016). This protocol is divided into four main steps: 1) stool samples are pulverized to evenly distribute microbial matter; 2) stool is enriched for virus-like particles and DNA is extracted by phenol-chloroform; 3) purified ...

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Isolation of Outer Membrane Vesicles from Phytopathogenic Xanthomonas campestris pv. campestris Authors: Gideon Mordukhovich and Ofir Bahar, date: 03/05/2017, view: 2795, Q&A: 1

Gram-negative bacteria naturally release outer membrane vesicles (OMVs) to the surrounding environment. OMVs contribute to multiple processes, such as cell-cell communication, delivery of enzymes and toxins, resistance to environmental stresses and pathogenesis. Little is known about OMVs produced by plant-pathogenic bacteria, and their ...

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Transient Transfection-based Fusion Assay for Viral Proteins Authors: Melina Vallbracht, Christina Schröter, Barbara G. Klupp and Thomas C. Mettenleiter, date: 03/05/2017, view: 3026, Q&A: 1

Membrane fusion is vital for entry of enveloped viruses into host cells as well as for direct viral cell-to-cell spread. To understand the fusion mechanism in more detail, we use an infection free system whereby fusion can be induced by a minimal set of the alphaherpesvirus pseudorabies virus (PrV) glycoproteins gB, gH and gL. Here, we describe an ...

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Chitin Extraction and Content Measurement in Magnaporthe oryzae Authors: Xinyu Liu and Zhengguang Zhang, date: 03/05/2017, view: 2418, Q&A: 0

Chitin is a linear polysaccharide composed of β (1→4)-linked N-acetylglucosamine (GlcNAc) residues. In fungi, chitin is an important component of the cell wall. Here, we provide a protocol to measure the chitin content of fungal cells using Magnaporthe oryzae as an example.

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Determination of Elemental Concentrations in Lichens Using ICP-AES/MS Authors: Liang-Cheng Zhao, Li Wang, Yun-Jun Jiang, Yan-Qiao Hu, Chong-Ying Xu, Lei Wang, Xing Li , Li Wei, Xiu-Ping Guo, Ai-Qin Liu and Hua-Jie Liu, date: 03/05/2017, view: 2406, Q&A: 0

Lichens are good biomonitors for air pollution because of their high enrichment capability of atmospheric chemical elements. To monitor atmospheric element deposition using lichens, it is important to obtain information on the multi-element concentrations in lichen thalli. Because of serious air pollution, elemental concentrations in thalli of ...

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Neuroscience

Extracellular Axon Stimulation Authors: Carola Städele, Margaret Louise DeMaegd and Wolfgang Stein, date: 03/05/2017, view: 3251, Q&A: 0

This is a detailed protocol explaining how to perform extracellular axon stimulations as described in Städele and Stein, 2016. The ability to stimulate and record action potentials is essential to electrophysiological examinations of neuronal function. Extracellular stimulation of axons traveling in fiber bundles (nerves) is a classical technique ...

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Axonal Conduction Velocity Measurement Authors: Margaret Louise DeMaegd, Carola Städele and Wolfgang Stein, date: 03/05/2017, view: 2331, Q&A: 0

Action potential conduction velocity is the speed at which an action potential (AP) propagates along an axon. Measuring AP conduction velocity is instrumental in determining neuron health, function, and computational capability, as well as in determining short-term dynamics of neuronal communication and AP initiation (Ballo and Bucher, 2009; ...

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Olfactory Avoidance Test (Mouse) Authors: Hiroo Takahashi and Akio Tsuboi, date: 03/05/2017, view: 2277, Q&A: 0

In mice, olfaction plays a pivotal role for the various behaviors, such as feeding, mating, nursing and avoidance. Behavioral tests that analyze abilities of odor detection and recognition using genetically modified mice reveal the contribution of target genes to the olfactory processing. Here, we describe the olfactory avoidance test to ...

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Olfactory Habituation-dishabituation Test (Mouse) Authors: Hiroo Takahashi and Akio Tsuboi, date: 03/05/2017, view: 2247, Q&A: 0

Olfaction plays a fundamental role in the various behaviors such as feeding, mating, nursing, and avoidance in mice. Behavioral tests that characterize abilities of odor detection and recognition using genetically modified mice reveal the contribution of target genes to the olfactory processing. Here, we describe the olfactory ...

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Thinned-skulled Cranial Window Preparation (Mice) Authors: Lifeng Zhang, Bo Liang, Yun Li and Da-Ting Lin, date: 03/05/2017, view: 2369, Q&A: 0

Imaging structural plasticity or activity of neurons in the brain circuit will facilitate understanding the neural mechanisms underlying animal behavior. Here we describe a modified procedure, the polished and reinforced thinned-skull cranial window preparation, by which we can image dendrites and spines in mouse layer I cortex for weeks (Zhang et ...

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Plant Science

Direct Visualization and Quantification of the Actin Nucleation and Elongation Events in vitro by TIRF Microscopy Authors: Yuxiang Jiang and Shanjin Huang, date: 03/05/2017, view: 2509, Q&A: 0

Total internal reflection fluorescence (TIRF) microscopy is a powerful tool for visualizing the dynamics of actin filaments at single-filament resolution in vitro. Thanks to the development of various fluorescent probes, we can easily monitor all kinds of events associated with actin dynamics, including nucleation, elongation, bundling, ...

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Acetyl Bromide Soluble Lignin (ABSL) Assay for Total Lignin Quantification from Plant Biomass Authors: William J. Barnes and Charles T. Anderson, date: 03/05/2017, view: 3981, Q&A: 0

Lignin is the second most abundant biopolymer on Earth, providing plants with mechanical support in secondary cell walls and defense against abiotic and biotic stresses. However, lignin also acts as a barrier to biomass saccharification for biofuel generation (Carroll and Somerville, 2009; Zhao and Dixon, 2011; Wang et al., 2013). For ...

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Laser Scanning Confocal Microcopy for Arabidopsis Epidermal, Mesophyll, and Vascular Parenchyma Cells Authors: Christian Elowsky, Yashitola Wamboldt and Sally Mackenzie, date: 03/05/2017, view: 3359, Q&A: 0

Investigation of protein targeting to plastids in plants by confocal laser scanning microscopy (CLSM) can be complicated by numerous sources of artifact, ranging from misinterpretations from in vivo protein over-expression, false fluorescence in cells under stress, and organellar mis-identification. Our studies have focused on the ...

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Knock-in Blunt Ligation Utilizing CRISPR/Cas9 Authors: Jonathan M. Geisinger and Michele P. Calos, date: 03/05/2017, view: 2978, Q&A: 0

The incorporation of the CRISPR/Cas9 bacterial immune system into the genetic engineering toolbox has led to the development of several new methods for genome manipulation (Auer et al., 2014; Byrne et al., 2015). We took advantage of the ability of Cas9 to generate blunt-ended double-strand breaks (Jinek et al., 2012) to ...

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Multiplexed GuideRNA-expression to Efficiently Mutagenize Multiple Loci in Arabidopsis by CRISPR-Cas9 Authors: Julia Schumacher, Kerstin Kaufmann and Wenhao Yan, date: 03/05/2017, view: 3618, Q&A: 0

Since the discovery of the CRISPR (clustered regularly interspaced short palindromic repeats)-associated protein (Cas) as an efficient tool for genome editing in plants (Li et al., 2013; Shan et al., 2013; Nekrasov et al., 2013), a large variety of applications, such as gene knock-out, knock-in or transcriptional ...

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Surface Inoculation and Quantification of Pseudomonas syringae Population in the Arabidopsis Leaf Apoplast Authors: Cristián Jacob, Shweta Panchal and Maeli Melotto, date: 03/05/2017, view: 3323, Q&A: 1

Bacterial pathogens must enter the plant tissue in order to cause a successful infection. Foliar bacterial pathogens that are not able to directly penetrate the plant epidermis rely on wounds or natural openings to internalize leaves. This protocol describes a procedure to estimate the population size of Pseudomonas syringae in the leaf ...

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In Gel Kinase Assay Authors: Gaston A. Pizzio and Pedro L. Rodriguez, date: 03/05/2017, view: 4092, Q&A: 0

Proper spatiotemporal regulation of protein phosphorylation in cells and tissues is required for normal development and homeostasis. We present the protocol ‘In Gel Kinase Assay’, which is useful for protein kinase activity measurements from crude protein extracts. We have successfully used ‘In Gel Kinase Assay’ protocol to show that the ...

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Stem Cell

Isolation and Primary Culture of Adult Human Adipose-derived Stromal/Stem Cells Authors: Robert B. Jones, Amy L. Strong, Jeffrey M. Gimble and Bruce A. Bunnell, date: 03/05/2017, view: 3631, Q&A: 0

Adipose-derived stromal/stem cells (ASCs) are multipotent cells that can be isolated from adipose tissue. Studies have shown that cells have the capacity to self-renew and differentiate into adipocyte, chondrocyte, myocyte, and osteoblast lineages. Thus, significant interest regarding their use for regenerative purposes to restore aging or damaged ...

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Reprogram Murine Epiblast Stem Cells by Epigenetic Inhibitors Authors: Hui Zhang and Yali Dou, date: 03/05/2017, view: 2104, Q&A: 0

Pluripotent stem cells in the naïve state are highly useful in regenerative medicine and tissue engineering. A robust reprogramming of the primed murine Epiblast Stem Cells (EpiSCs) to naïve pluripotency is feasible via chemical-only approach. This protocol described a method to reprogram murine EpiSCs by MM-401 treatment, which blocks histone ...

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