Volume: 7 Issue: 11 —BIO-PROTOCOL

Biochemistry

Heavy Metal Stress Assay of Caenorhabditis elegans Authors: Strahil Iv. Pastuhov, Tatsuhiro Shimizu and Naoki Hisamoto, date: 06/05/2017, view: 7397, Q&A: 0

Organisms have developed many protective systems to reduce the toxicity from heavy metals. The nematode Caenorhabditis elegans has been widely used to determine the protective mechanisms against heavy metals. Responses against heavy metals can be monitored by expression of reporter genes, while sensitivity can be determined by quantifying ...

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Cancer Biology

DNA Fiber Assay upon Treatment with Ultraviolet Radiations Authors: Alfano Luigi, Antonio Giordano and Francesca Pentimalli, date: 06/05/2017, view: 13131, Q&A: 1

Genome stability is continuously challenged by a wide range of DNA damaging factors. To promote a correct DNA repair and cell survival, cells orchestrate a coordinated and finely tuned cascade of events collectively known as the DNA Damage Response (DDR). Ultra Violet (UV) rays are among the main environmental sources of DNA damage and a well ...

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Fluorometric Estimation of Glutathione in Cultured Microglial Cell Lysate Authors: Vikas Singh, Ruchi Gera, Mahaveer Prasad Purohit, Satyakam Patnaik and Debabrata Ghosh, date: 06/05/2017, view: 8469, Q&A: 6

Glutathione is one of the major antioxidant defense components present in cells. It is predominantly present as reduced glutathione (GSH) and converted into oxidized glutathione (GSSG) while reducing the free radicals like hydroxyl ions (OH^-). For the measurement of GSH and GSSG, o-phthalaldehyde (OPT) has been used as a fluorescent ...

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Single-molecule Analysis of DNA Replication Dynamics in Budding Yeast and Human Cells by DNA Combing Authors: Hélène Tourrière, Julie Saksouk, Armelle Lengronne and Philippe Pasero , date: 06/05/2017, view: 9719, Q&A: 0

The DNA combing method allows the analysis of DNA replication at the level of individual DNA molecules stretched along silane-coated glass coverslips. Before DNA extraction, ongoing DNA synthesis is labeled with halogenated analogues of thymidine. Replication tracks are visualized by immunofluorescence using specific antibodies. Unlike biochemical ...

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Intracaecal Orthotopic Colorectal Cancer Xenograft Mouse Model Authors: Hsin-Wei Liao and Mien-Chie Hung, date: 06/05/2017, view: 9125, Q&A: 1

The host microenvironment plays a prominent role in tumor growth, angiogenesis, invasion, metastasis, and response to therapy. Orthotopic tumor model mimics the natural environment of tumor development and provides an effective approach to investigate tumor pathophysiology and develop therapeutic strategies. This protocol describes the technique ...

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Pituitary Isograft Transplantation in Mice Authors: Chance Walker, Yan Hong, Frances Kittrell, Daniel Medina, David Edwards and Fariba Behbod, date: 06/05/2017, view: 5559, Q&A: 0

The mouse pituitary isograft is a technique developed to administer persistent hormone stimulation, thereby increasing cellular proliferation in the mammary tissue (Christov et al., 1993). The pituitary isograft procedure was first described in ‘Induction of Mammary Cancer in Mice without the Mammary Tumor Agent by Isografts of ...

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Whole Mammary Gland Transplantation in Mice Protocol Authors: Hayley Hansford, Yan Hong, Frances Kittrell, Daniel Medina and Fariba Behbod, date: 06/05/2017, view: 5719, Q&A: 0

Whole Mammary Gland Transplantation involves transplanting an excised mammary gland into another, more suitable host. This method can be used to extend the life of a mammary gland past the mouse’s life span by transplanting the mammary gland of an older mouse into a young healthy mouse. As you can see in the video below (Video 1), by attaching it ...

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Targeted Nucleotide Substitution in Mammalian Cell by Target-AID Authors: Takayuki Arazoe, Keiji Nishida and Akihiko Kondo, date: 06/05/2017, view: 10672, Q&A: 0

Programmable RNA-guided nucleases based on CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated protein) systems have been applied to various type of cells as powerful genome editing tools. By using activation-induced cytidine deaminase (AID) in place of the nuclease activity of the CRISPR/Cas9 system, we have ...

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Cell Biology

Flow Cytometric Analysis of HIV-1 Transcriptional Activity in Response to shRNA Knockdown in A2 and A72 J-Lat Cell Lines Authors: Daniela Boehm and Melanie Ott, date: 06/05/2017, view: 6560, Q&A: 0

The main obstacle to eradicating HIV-1 from patients is post-integration latency (Finzi et al., 1999). Antiretroviral treatments target only actively replicating virus, while latent infections that have low or no transcriptional activity remain untreated (Sedaghat et al., 2007). To eliminate viral reservoirs, one strategy focuses ...

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Generation of Mutant Pigs by Direct Pronuclear Microinjection of CRISPR/Cas9 Plasmid Vectors Authors: Chin-kai Chuang, Ching-Fu Tu and Chien-Hong Chen, date: 06/05/2017, view: 7858, Q&A: 0

A set of Cas9 and single guide CRISPR RNA expression vectors was constructed. Only a very simple procedure was needed to prepare specific single-guide RNA expression vectors with high target accuracy. Since the de novo zygotic transcription had been detected in mouse embryo at the 1-cell stage, the plasmid DNA vectors encoding Cas9 and ...

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Immunology

Isolation and Infection of Drosophila Primary Hemocytes Authors: Charles Tracy and Helmut Krämer, date: 06/05/2017, view: 6992, Q&A: 0

Phagocytosis of invading pathogens and their subsequent clearance in lysosomes is important for organismal fitness. We have devised the following protocol to extract phagocytic hemocytes from wild-type and mutant Drosophila larvae and infect the isolated hemocytes with GFP-labeled E. coli to measure the rate of phagocytosis and ...

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ELISPOT Assay to Measure Peptide-specific IFN-γ Production Authors: Michelle N. Wykes and Laurent Renia, date: 06/05/2017, view: 14390, Q&A: 0

Interferon-gamma (IFN-γ) is crucial for immunity against intracellular pathogens and for tumor control. It is produced predominantly by natural killer (NK) and natural killer T cells (NKT) as well as by antigen-specific Th1 CD4⁺ and CD8⁺ effector T cells. When investigating immune responses against pathogens and cancer cells, ...

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In vitro Antigen-presentation Assay for Self- and Microbial-derived Antigens Author: Laura Campisi, date: 06/05/2017, view: 12219, Q&A: 0

Antigen presenting cells (APC) are able to process and present to T cells antigens from different origins. This mechanism is highly regulated, in particular by Patter Recognition Receptor (PRR) signals. Here, I detail a protocol designed to assess in vitro the capacity of APC to present antigens derived from bacteria, apoptotic and ...

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Microbiology

Fluorescently Labelled Aerolysin (FLAER) Labelling of Candida albicans Cells Authors: Sneh Lata Singh and Sneha Sudha Komath, date: 06/05/2017, view: 6617, Q&A: 0

In this protocol we describe a nonradiolabelled labelling of GPI anchor in Candida albicans. The method uses a fluorescent probe to bind specifically to GPI anchors so that the level of GPI-anchored proteins at the cell surface can be measured. The labelling does not need permeabilization of cells and can be carried out in vivo.

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Ex vivo Model of Human Aortic Valve Bacterial Colonization Authors: Alejandro Avilés-Reyes, Irlan Almeida Freires, Pedro Luiz Rosalen, José A. Lemos and Jacqueline Abranches, date: 06/05/2017, view: 4880, Q&A: 0

The interaction of pathogens with host tissues is a key step towards successful colonization and establishment of an infection. During bacteremia, pathogens can virtually reach all organs in the human body (e.g., heart, kidney, spleen) but host immunity, blood flow and tissue integrity generally prevents bacterial colonization. Yet, ...

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Protein Localization in the Cyanobacterium Anabaena sp. PCC7120 Using Immunofluorescence Labeling Authors: Carla Trigo, Derly Andrade and Mónica Vásquez, date: 06/05/2017, view: 5605, Q&A: 0

Techniques such as immunoflorescence are widely used to determine subcellular distribution of proteins. Here we report on a method to immunolocalize proteins in Anabaena sp. PCC7120 with fluorophore-conjugated antibodies by fluorescence microscopy. This method improves the permeabilization of cyanobacterial cells and minimizes the ...

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Induction and Quantification of Patulin Production in Penicillium Species Authors: Yong Chen, Boqiang Li, Zhanquan Zhang and Shiping Tian, date: 06/05/2017, view: 5974, Q&A: 0

Patulin, a worldwide regulated mycotoxin, is primarily produced by Penicillium and Aspergillus species during fruit spoilage. Patulin contamination is a great concern with regard to human health because exposure of the mycotoxin can result in severe acute and chronic toxicity, including neurotoxic, mutagenic, and immunotoxic ...

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Phototaxis Assays of Synechocystis sp. PCC 6803 at Macroscopic and Microscopic Scales Authors: Annik Jakob, Nils Schuergers and Annegret Wilde, date: 06/05/2017, view: 6231, Q&A: 0

Phototaxis is a mechanism that allows cyanobacteria to respond to fluctuations in the quality and quantity of illumination by moving either towards or away from a light source. Phototactic movement on low concentration agar or agarose plates can be analyzed at macroscopic and microscopic scales representing group behavior and single cell motility, ...

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A Reliable Assay to Evaluate the Virulence of Aspergillus nidulans Using the Alternative Animal Model Galleria mellonella (Lepidoptera) Authors: Caroline Mota Fernandes, Fernanda Lopes Fonseca, Gustavo Henrique Goldman, Marcos Dias Pereira and Eleonora Kurtenbach, date: 06/05/2017, view: 7284, Q&A: 0

The greater wax moth Galleria mellonella has emerged as an effective heterologous host to study fungal pathogenesis and the efficacy of promising antifungal drugs (Mylonakis et al., 2005; Li et al., 2013). Here, a methodology describing the Aspergillus nidulans infection in G. mellonella larvae, along ...

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Expression and Purification of the Cas10-Csm Complex from Staphylococci Authors: Lucy Chou-Zheng and Asma Hatoum-Aslan, date: 06/05/2017, view: 7273, Q&A: 0

CRISPR-Cas (Clustered regularly interspaced short palindromic repeats-CRISPR-associated proteins) is a class of prokaryotic immune systems that degrade foreign nucleic acids in a sequence-specific manner. These systems rely upon ribonucleoprotein complexes composed of Cas nucleases and small CRISPR RNAs (crRNAs). Staphylococcus epidermidis ...

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Molecular Biology

Formation of Minimised Hairpin Template-transcribing Dumbbell Vectors for Small RNA Expression Authors: Xiaoou Jiang and Volker Patzel, date: 06/05/2017, view: 6473, Q&A: 0

A major barrier for using non-viral vectors for gene therapy is the short duration of transgene expression in postmitotic tissues. Previous studies showed transgene expression from conventional plasmid fell to sub-therapeutic level shortly after delivery even though the vector DNA was retained, suggesting transcription was silenced in vivo ...

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Chromatin Immunoprecipitation Experiments from Whole Drosophila Embryos or Larval Imaginal Discs Authors: Vincent Loubiere, Anna Delest, Bernd Schuettengruber, Anne-Marie Martinez and Giacomo Cavalli, date: 06/05/2017, view: 9013, Q&A: 1

Chromatin Immunoprecipitation coupled either to qPCR (qChIP) or high-throughput sequencing (ChIP-Seq) has been extensively used in the last decades to identify the DNA binding sites of transcription factors or the localization of various histone marks along the genome. The ChIP experiment generally includes 7 steps: collection of biological ...

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A Protocol for Production of Mutant Mice Using Chemically Synthesized crRNA/tracrRNA with Cas9 Nickase and FokI-dCas9 Authors: Satoshi Hara, Miho Terao and Shuji Takada, date: 06/05/2017, view: 7507, Q&A: 0

The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system is the most widely used genome editing tool. A common CRISPR/Cas9 system consists of two components: a single-guide RNA (sgRNA) and Cas9. Both components are required for the introduction of a double-strand break (DSB) at a specific ...

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Neuroscience

Kinetic Lactate Dehydrogenase Assay for Detection of Cell Damage in Primary Neuronal Cell Cultures Authors: Dorette Freyer and Christoph Harms, date: 06/05/2017, view: 8058, Q&A: 0

The aim of many in vitro models of acute or chronic degenerative disorders in the neurobiology field is the assessment of survival or damage of neuronal cells. Damage of cells is associated with loss of outer cell membrane integrity and leakage of cytoplasmic cellular proteins. Therefore, activity assays of cytoplasmic enzymes in ...

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The Repeated Flurothyl Seizure Model in Mice Author: Russell J. Ferland, date: 06/05/2017, view: 5600, Q&A: 0

Development of spontaneous seizures is the hallmark of human epilepsy. There is a critical need for new epilepsy models in order to elucidate mechanisms responsible for leading to the development of spontaneous seizures and for testing new anti-epileptic compounds. Moreover, rodent models of epilepsy have clearly demonstrated that there are two ...

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Plant Science

Photometric Assays for Chloroplast Movement Responses to Blue Light Authors: Halina Gabryś, Agnieszka Katarzyna Banaś, Pawel Hermanowicz, Weronika Krzeszowiec, Sebastian Leśniewski, Justyna Łabuz and Olga Sztatelman, date: 06/05/2017, view: 5496, Q&A: 0

Assessment of chloroplast movements by measuring changes in leaf transmittance is discussed, with special reference to the conditions necessary for reliable estimation of blue light–activated chloroplast responses.

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Protocol for Enrichment of the Membrane Proteome of Mature Tomato Pollen Authors: Puneet Paul, Palak Chaturvedi, Anida Mesihovic, Arindam Ghatak, Wolfram Weckwerth and Enrico Schleiff, date: 06/05/2017, view: 5493, Q&A: 0

We established and elaborated on a method to enrich the membrane proteome of mature pollen from economically relevant crop using the example of Solanum lycopersicum (tomato). To isolate the pollen protein fraction enriched in membrane proteins, a high salt concentration (750 mM of sodium chloride) was used. The membrane protein-enriched ...

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Determining Genome Size from Spores of Seedless Vascular Plants Authors: Li-Yaung Kuo and Yao-Moan Huang , date: 06/05/2017, view: 6040, Q&A: 0

Seedless vascular plants, including ferns and lycophytes, produce spores to initiate the gametophyte stage and to complete sexual reproduction. Approximately 10% of them are apomictic through the production of genomic unreduced spores. Being able to measure the spore nuclear DNA content is therefore important to infer their reproduction mode. Here ...

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Whole-seed Immunolabeling of Arabidopsis Mucilage Polysaccharides Author: Cătălin Voiniciuc, date: 06/05/2017, view: 4981, Q&A: 0

In addition to synthesizing and secreting copious amounts of pectic polymers (Young et al., 2008), Arabidopsis thaliana seed coat epidermal cells produce small amounts of cellulose and hemicelluloses typical of secondary cell walls (Voiniciuc et al., 2015c). These components are intricately linked and are released as a ...

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DNA-free Genome Editing of Chlamydomonas reinhardtii Using CRISPR and Subsequent Mutant Analysis Authors: Jihyeon Yu, Kwangryul Baek, EonSeon Jin and Sangsu Bae, date: 06/05/2017, view: 10005, Q&A: 0

We successfully introduced targeted knock-out of gene of interest in Chlamydomonas reinhardtii by using DNA-free CRISPR. In this protocol, the detailed procedures of an entire workflow cover from the initial target selection of CRISPR to the mutant analysis using next generation sequencing (NGS) technology. Furthermore, we introduce a ...

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Stem Cell

Functional Analysis of Connexin Channels in Cultured Cells by Neurobiotin Injection and Visualization Authors: Philipp Wörsdörfer and Klaus Willecke, date: 06/05/2017, view: 5957, Q&A: 0

Functional gap junction channels between neighboring cells can be assessed by microinjection of low molecular weight tracer substances into cultured cells. The extent of direct intercellular communication can be precisely quantified by this method. This protocol describes the iontophoretic injection and visualisation of Neurobiotin into cultured ...

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Past Issues in 2017

Volume: 7 Issue: 11

Biochemistry

Cancer Biology

Cell Biology

Immunology

Microbiology

Molecular Biology

Neuroscience

Plant Science

Stem Cell