• Volume 9, 2019
  • Volume 8, 2018
  • Volume 7, 2017
  • Volume 6, 2016
  • Volume 5, 2015
  • Volume 4, 2014
  • Volume 3, 2013
  • Volume 2, 2012
  • Volume 1, 2011

Biochemistry

Quantitation of Cytochromes b559, b6, and f, and the Core Component of Photosystem I P700 in Cyanobacterial Cells Authors:  Motohide Aoki, Mikio Tsuzuki and Norihiro Sato, date: 11/05/2016, view: 4216, Q&A: 0
Cytochrome (Cyt) b559, an important and essential core component of photosystem II in the photosynthetic electron transport system, is a heme-bridged heterodimer protein composed of an alpha subunit (PsbE) and a beta subunit (PsbE), and its reduced form has an absorption maximum in the α-band at 559 nm. The ...
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Cancer Biology

Sulforhodamine B (SRB) Assay in Cell Culture to Investigate Cell Proliferation Authors:  Esteban A. Orellana and Andrea L. Kasinski, date: 11/05/2016, view: 29678, Q&A: 0
The SRB assay has been used since its development in 1990 (Skehan et al., 1990) to inexpensively conduct various screening assays to investigate cytotoxicity in cell based studies (Vichai and Kirtikara, 2006). This method relies on the property of SRB, which binds stoichiometrically to proteins under mild acidic conditions and then can be ...
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Fat Turnover Assay in Drosophila Authors:  Subhash D. Katewa and Pankaj Kapahi, date: 11/05/2016, view: 6462, Q&A: 0
Like all animals, Drosophila shows robust fat (triglyceride) turnover, i.e., they synthesize, store and utilize triglyceride for their daily metabolic needs. The protocol describes a simple assay to measure this turnover of triglycerides in Drosophila.
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Phagocytosis Assay to Measure Uptake of Necroptotic Cancer Cells by BMDCs Authors:  Tania Løve Aaes, Dmitri V. Krysko and Peter Vandenabeele, date: 11/05/2016, view: 6367, Q&A: 0
This protocol is a flow cytometry-based method to measure the phagocytosis efficiency of necroptotic target cells by bone marrow-derived dendritic cells (BMDCs) in vitro (Aaes et al., 2016). The method is a slightly modified and updated version of the protocols used in previously published papers (Krysko et al., 2006; ...
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Cell Biology

Determination of the Glycolysis and Lipogenesis in Culture of Hepatocytes Authors:  Pierre-Damien Denechaud and Luis Fajas, date: 11/05/2016, view: 6926, Q&A: 0
Metabolic flux analyses are needed to provide insights into metabolic regulation that occurs in cells. The current protocol describes fast and reproducible methods for determining glycolysis and de novo lipogenesis of hepatocytes. Primary culture of hepatocytes is an ‘in vitro’ model useful to study liver glucose and lipid ...
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Immunology

Reconstruction of the Mouse Inflammasome System in HEK293T Cells Authors:  Hexin Shi, Anne Murray and Bruce Beutler, date: 11/05/2016, view: 5363, Q&A: 0
The NLRP3 (NLR family, Pyrin domain containing 3) inflammasome is a multiprotein complex comprised of NLRP3, pro-caspase-1, the adaptor protein apoptosis-associated speck-like protein containing a CARD (ASC), and the protein kinase NIMA related kinase 7 (NEK7) (Shi et al., 2016; He et al., 2016; Schmid-Burgk et al., ...
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Microbiology

Determination of (p)ppGpp Levels During Stringent Response in Streptomyces coelicolor by Thin Layer Chromatography Authors:  Smitha Sivapragasam and Anne Grove, date: 11/05/2016, view: 5670, Q&A: 0
The stringent response in bacteria is a stress response that is mediated by the signaling molecules guanosine tetraphosphate and pentaphosphate [(p)ppGpp], alarmones that are also directly related to virulence. Therefore, determination of (p)ppGpp levels is crucial for studying the stringent response. The protocol here outlines in a step-wise ...
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Sequencing of Ebola Virus Genomes Using Nanopore Technology Author:  Thomas Hoenen, date: 11/05/2016, view: 6150, Q&A: 0
Sequencing of virus genomes during disease outbreaks can provide valuable information for diagnostics, epidemiology, and evaluation of potential countermeasures. However, particularly in remote areas logistical and technical challenges can be significant. Nanopore sequencing provides an alternative to classical Sanger and next-generation ...
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Molecular Biology

Identification of Methylated Deoxyadenosines in Genomic DNA by dA6m DNA Immunoprecipitation Authors:  Magdalena J. Koziol, Charles R. Bradshaw, George E. Allen, Ana S. H. Costa and Christian Frezza, date: 11/05/2016, view: 4989, Q&A: 0
dA6m DNA immunoprecipitation followed by deep sequencing (DIP-Seq) is a key tool in identifying and studying the genome-wide distribution of N6-methyldeoxyadenosine (dA6m). The precise function of this novel DNA modification remains to be fully elucidated, but it is known to be absent from transcriptional start ...
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Neuroscience

Microglial Phagocytosis Assay Authors:  Hong Lian, Ethan Roy and Hui Zheng, date: 11/05/2016, view: 9236, Q&A: 0
Phagocytosis is essential for microglial clearance of apoptotic cells, extracellular protein aggregates, and infectious bacteria in the central nervous system (CNS). While the preparation of primary microglial culture has been described elsewhere, this protocol describes the microglial phagocytosis experimental procedure and the subsequent ...
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Protocol for Primary Microglial Culture Preparation Authors:  Hong Lian, Ethan Roy and Hui Zheng, date: 11/05/2016, view: 11544, Q&A: 0
Primary microglia, in either mono-culture or co-culture with neurons or astrocytes, are a powerful tool for studying mechanisms underlying microglial inflammatory responses and cell type-specific interactions in the central nervous system (CNS). This protocol provides the details of how to prepare high purity primary microglia from newborn mouse ...
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Apparatus and General Methods for Exposing Rats to Audiogenic Stress Author:  Serge Campeau, date: 11/05/2016, view: 3670, Q&A: 0
Most organisms react innately to the sudden onset of environmental stimulation. Audiogenic or loud noise in rodents provides an effective threatening signal to study the central nervous circuits responsible for the elaboration of various responses typically elicited by threatening/stressful environmental stimulation. Audiogenic stress offers many ...
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Plant Science

Metabolite Profiling of Mature Arabidopsis thaliana Seeds Using Gas Chromatography-Mass Spectrometry (GC-MS) Authors:  Hagai Cohen, Ifat Matityahu and Rachel Amir, date: 11/05/2016, view: 6100, Q&A: 0
Metabolite profiling using gas chromatography-mass spectrometry (GC-MS) permits the annotation and quantification of a relatively wide variety of metabolites, covering a wide range of biochemical groups of metabolites. Lisec et al. (2006) established a method for GC-MS profiling in plants. Based on this protocol, we provide here a ...
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Determination of Recombinant Mannitol-1-phosphate Dehydrogenase Activity from Ectocarpus sp. Authors:  Agnès Groisillier and Thierry Tonon, date: 11/05/2016, view: 4380, Q&A: 0
Brown algae belong to a phylogenetic lineage distantly related to green plants and animals, and are found predominantly, but not exclusively, in the intertidal zone, a harsh and frequently changing environment. Because of their unique evolutionary history and of their habitat, brown algae feature several peculiarities in their metabolism. One of ...
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Cytohistological Analyses of Mega-sporogenesis and Gametogenesis in Ovules of Limonium spp. Authors:  Ana S. Róis and Ana D. Caperta, date: 11/05/2016, view: 3806, Q&A: 0
Limonium spp. are known to have sexual and apomixis (asexual reproduction through seeds) reproductive modes. Here, we present dissection protocol developed for ovules of Limonium spp. using differential interference contrast (DIC) microscopy. This protocol permits better handling of ovules and offers certain advantages over ...
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A Ribosome Footprinting Protocol for Plants Authors:  Catharina Merchante, Qiwen Hu, Steffen Heber, Jose Alonso and Anna N. Stepanova, date: 11/05/2016, view: 7448, Q&A: 0
Ribosome footprinting, or Ribo-seq, has revolutionized the studies of translation. It was originally developed for yeast and mammalian cells in culture (Ingolia et al., 2009). Herein, we describe a plant-optimized hands-on ribosome footprinting protocol derived from previously published procedures of polysome isolation (Ingolia et al ...
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Putrescine Biosynthesis Inhibition in Tomato by DFMA and DFMO Treatment Authors:  Emma Fernández-Crespo, Ana Isabel González-Hernández, Loredana Scalschi, Eugenio Llorens, Pilar García-Agustín and Gemma Camañes, date: 11/05/2016, view: 4257, Q&A: 0
This protocol can be used to inhibit the biosynthesis of polyamines, specifically putrescine, in tomato plants grown with NH4+ as a solely N source. In general, polyamines are positively charged small metabolites implicated in physiological processes, including organogenesis, embryogenesis, floral initiation and development, ...
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Hydrogen Peroxide Measurement in Arabidopsis Root Tissue Using Amplex Red Authors:  Tzvetina Brumbarova, Cham Thi Tuyet Le and Petra Bauer, date: 11/05/2016, view: 8065, Q&A: 0
This protocol describes the measurement of hydrogen peroxide (H2O2) content in Arabidopsis root tissue by using the Amplex® Red Hydrogen Peroxide/Peroxidase Assay Kit. When root tissue is disrupted and resuspended in phosphate buffer, H2O2 is released from the cells. The obtained root ...
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PEA-CLARITY: Three Dimensional (3D) Molecular Imaging of Whole Plant Organs Authors:  William M. Palmer, Antony P. Martin, Jamie R. Flynn, Stephanie Reed, Rosemary White, Robert T. Furbank and Christopher P. L Grof, date: 11/05/2016, view: 4416, Q&A: 0
Here we report the adaptation of the CLARITY technique to plant tissues with addition of enzymatic degradation to improve optical clearing and facilitate antibody probe penetration. Plant-Enzyme-Assisted (PEA)-CLARITY, has allowed deep optical visualisation of stains, expressed fluorescent proteins and IgG-antibodies in tobacco and Arabidopsis ...
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Stem Cell

Allogeneic Transplantation of Testicular Hyperplasia in rag1 Mutant Zebrafish Authors:  Toshihiro Kawasaki and Noriyoshi Sakai, date: 11/05/2016, view: 4303, Q&A: 0
Allogeneic organ transplantation is a powerful tool for clinical and basic research studies. However, the graft is often rejected by the host organism. Here, we describe a protocol that uses immunodeficient rag1 mutant zebrafish. These zebrafish escaped rejection, which made it possible to successfully transplant fragments of an ...
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