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Past Issues in Bio-protocol

Volume: 7 Issue: 18September 20, 2017

  • All fields (15)
  • Biochemistry (3)
  • Cancer Biology (1)
  • Cell Biology (1)
  • Immunology (3)
  • Microbiology (2)
  • Neuroscience (3)
  • Plant Science (2)

Biochemistry

In vitro Assays for Eukaryotic Leading/Lagging Strand DNA Replication Authors:  Grant Schauer, Jeff Finkelstein and Mike O’Donnell, date: 09/20/2017, view: 502, Q&A: 0
The eukaryotic replisome is a multiprotein complex that duplicates DNA. The replisome is sculpted to couple continuous leading strand synthesis with discontinuous lagging strand synthesis, primarily carried out by DNA polymerases ε and δ, respectively, along with helicases, polymerase α-primase, DNA sliding clamps, clamp loaders and many other ...
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Lipidomic Analysis of Caenorhabditis elegans Embryos Authors:  Hung-Chi Yang, Cheng-Yu Hung, Yi-Yun Pan, Szecheng J Lo and Daniel Tsun-Yee Chiu, date: 09/20/2017, view: 415, Q&A: 0
Metabolomic is an emerging field of system biology. Lipidomic, a branch of metabolomic, aims to characterize lipophilic metabolites in biological systems. Caenorhabditis elegans (C. elegans) is a genetically tractable and versatile animal model for novel discovery of lipid metabolism. In addition, C. elegans embryo is ...
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Protease Activity Assay in Fly Intestines Authors:  Marie-Paule Nawrot-Esposito, Rihab Loudhaief and Armel Gallet, date: 09/20/2017, view: 457, Q&A: 0
The intestine is a central organ required for the digestion of food, the absorption of nutrients and for fighting against aggressors ingested along with the food. Impairment of gut physiology following mucosal damages impacts its digestive capacities that consequently will affect growth, wellbeing or even survival of the individual. Hence, the ...
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Cancer Biology

Uptake Assays to Monitor Anthracyclines Entry into Mammalian Cells Authors:  Nicolas Brosseau, Emil Andreev and Dindial Ramotar, date: 09/20/2017, view: 320, Q&A: 0
Anthracyclines, such as doxorubicin and daunorubicin, are DNA damaging agents that autofluoresce and can be readily detected in cells. Herein, we developed suitable assays to quantify and localize daunorubicin in mammalian cells. These assays can be exploited to identify components that are involved in the uptake of anthracyclines.
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Cell Biology

Freeze-fracture-etching Electron Microscopy for Facile Analysis of Yeast Ultrastructure Authors:  Takuma Tsuji and Toyoshi Fujimoto, date: 09/20/2017, view: 449, Q&A: 0
We describe a streamlined method that enables the quick observation of yeast ultrastructure by electron microscopy (EM). Yeast cells are high-pressure frozen, freeze-fractured to cut across the cytoplasm, and freeze-etched to sublimate ice in the cytosol and the organelle lumen. The cellular structures delineated by these procedures are coated by ...
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Immunology

Phagocytosis Assay of Necroptotic Cells by Cardiac Myofibroblasts Authors:  Yuma Horii, Shoichi Matsuda, Kenji Watari, Akiomi Nagasaka, Hitoshi Kurose and Michio Nakaya, date: 09/20/2017, view: 407, Q&A: 0
In myocardial infarction (MI), a plenty of cardiomyocytes undergo necrosis and necroptosis due to the lack of oxygen and nutrients. The dead cardiomyocytes are promptly engulfed by phagocytes. When the dead cells are not engulfed, the noxious contents of the cells are released outside, and thus, induce inflammation, and obstruct the function of ...
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An Assay to Determine Phagocytosis of Apoptotic Cells by Cardiac Macrophages and Cardiac Myofibroblasts Authors:  Yuma Horii, Shoichi Matsuda, Kenji Watari, Akiomi Nagasaka, Hitoshi Kurose and Michio Nakaya, date: 09/20/2017, view: 310, Q&A: 0
In myocardial infarction (MI), a number of cardiomyocytes undergo apoptosis. These apoptotic cardiomyocytes are promptly engulfed by phagocytes. If the dead cells are not engulfed, their noxious contents are released outside, resulting in induction of inflammation. Therefore, the removal of these dead cells is necessary. However, the contribution ...
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Differentiation of Myeloid-derived Suppressor Cells from Murine Bone Marrow and Their Co-culture with Splenic Dendritic Cells Authors:  Giada Mondanelli and Claudia Volpi, date: 09/20/2017, view: 382, Q&A: 0
Myeloid-derived suppressor cells (MDSCs) possess the ability to suppress the immune response, and to amplify the regulatory properties of other immune cells, i.e., dendritic cells. Here we describe a protocol in which MDSCs were differentiated from murine bone marrow cells, and CD11c+ dendritic cells were purified from murine ...
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Microbiology

Drosophila Fecal Sampling Authors:  Christine Fink , Jakob von Frieling, Mirjam Knop and Thomas Roeder, date: 09/20/2017, view: 375, Q&A: 0
Fecal sampling is a non-invasive method which raises the possibility to study the development and the changes in the microbial community throughout different time points of a fly population or throughout different treatments. This method allows precise manipulation to trigger the fly’s physiology by nutritional interventions, bacterial infections ...
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Method for Multiplexing CRISPR/Cas9 in Saccharomyces cerevisiae Using Artificial Target DNA Sequences Authors:  Rachael M. Giersch and Gregory C. Finnigan, date: 09/20/2017, view: 564, Q&A: 0
Genome manipulation has become more accessible given the advent of the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) editing technology. The Cas9 endonuclease binds a single stranded (single guide) RNA (sgRNA) fragment that recruits the complex to a corresponding genomic target sequence where it induces a double stranded ...
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Neuroscience

Stereotaxic Adeno-associated Virus Injection and Cannula Implantation in the Dorsal Raphe Nucleus of Mice Authors:  Patrícia A. Correia, Sara Matias and Zachary F. Mainen, date: 09/20/2017, view: 417, Q&A: 0
Optogenetic methods are now widespread in neuroscience research. Here we present a detailed surgical procedure to inject adeno-associated viruses and implant optic fiber cannulas in the dorsal raphe nucleus (DRN) of living mice. Combined with transgenic mouse lines, this protocol allows specific targeting of serotonin-producing neurons in the ...
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Bioluminescence Monitoring of Neuronal Activity in Freely Moving Zebrafish Larvae Authors:  Steven Knafo, Andrew Prendergast, Olivier Thouvenin, Sophie Nunes Figueiredo and Claire Wyart, date: 09/20/2017, view: 378, Q&A: 0
The proof of concept for bioluminescence monitoring of neural activity in zebrafish with the genetically encoded calcium indicator GFP-aequorin has been previously described (Naumann et al., 2010) but challenges remain. First, bioluminescence signals originating from a single muscle fiber can constitute a major pitfall. Second, ...
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Preparation of Primary Cultures of Embryonic Rat Hippocampal and Cerebrocortical Neurons Authors:  Ivan L. Salazar, Miranda Mele, Margarida V. Caldeira, Rui O. Costa, Bárbara Correia, Simone Frisari and Carlos B. Duarte, date: 09/20/2017, view: 384, Q&A: 0
This protocol aims at standardizing the procedure to obtain primary cultures of hippocampal and cerebrocortical neurons for in vitro experiments. Cultures should be prepared from cells isolated during embryonic development when neuronal precursor cells are not yet fully differentiated. This helps increasing the quality and quantity of ...
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Plant Science

Detection of Protein S-nitrosothiols (SNOs) in Plant Samples on Diaminofluorescein (DAF) Gels Authors:  Marta Rodríguez-Ruiz, Paulo T. Mioto, José M. Palma and Francisco J. Corpas , date: 09/20/2017, view: 393, Q&A: 0
In plant cells, the analysis of protein S-nitrosothiols (SNOs) under physiological and adverse stress conditions is essential to understand the mechanisms of Nitric oxide (NO)-based signaling. We adapted a previously reported protocol for detecting protein SNOs in animal systems (King et al., 2005) for plant samples. Briefly, ...
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Isolation and Detection of the Chlorophyll Catabolite Hydroxylating Activity from Capsicum annuum Chromoplasts Authors:  Mareike Hauenstein and Stefan Hörtensteiner, date: 09/20/2017, view: 491, Q&A: 0
Hydroxylation of chlorophyll catabolites at the so-called C32 position (Hauenstein et al., 2016) is commonly found in all plant species analyzed to date. Here we describe an in vitro hydroxylation assay using Capsicum annuum chromoplast membranes as a source of the hydroxylating activity, which converts the ...
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