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Past Issues in Bio-protocol

Volume: 6 Issue: 22November 20, 2016

  • All fields (28)
  • Biochemistry (3)
  • Cancer Biology (3)
  • Cell Biology (1)
  • Immunology (5)
  • Microbiology (7)
  • Neuroscience (3)
  • Plant Science (4)
  • Stem Cell (2)

Biochemistry

Determining Efficiency and Selectivity of Lipid Extraction by Perturbing Agents from Model Membranes Authors:  Michel Lafleur and Alexandre Therrien, date: 11/20/2016, view: 1254, Q&A: 0
Several membrane-perturbing agents extract lipids from membranes and, in some cases, this lipid efflux is lipid specific. In order to gain a better description of this phenomenon and to detail the intermolecular interactions that are involved, a method has been developed to characterize the extent and the specificity of membrane-lipid extraction ...
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Determination of Rate of [3H-methyl]-choline Incorporation into Cellular Lipids and Non-lipid Metabolites Authors:  Tim Andrew Davies Smith and Su Myat Phyu, date: 11/20/2016, view: 1199, Q&A: 0
The choline-containing phospholipid, phosphatidylcholine (PtdCho) is the most common mammalian phospholipid found in cell membrane (Ide et al., 2013). It is also a component of intracellular signalling pathways (Cui and Houweling, 2002). Herein is described a measure of the rate of accumulation of choline by lipid soluble PtdCho and ...
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Ionization Properties of Phospholipids Determined by Zeta Potential Measurements Authors:  Murugappan Sathappa and Nathan N. Alder, date: 11/20/2016, view: 1842, Q&A: 0
Biological membranes are vital for diverse cellular functions such as maintaining cell and organelle structure, selective permeability, active transport, and signaling. The surface charge of the membrane bilayer plays a critical role in these myriad processes. For most biomembranes, the surface charge of anionic phospholipids contributes to the ...
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Cancer Biology

Establishment of Patient-Derived Xenografts in Mice Authors:  Dongkyoo Park, Dongsheng Wang, Guo Chen and Xingming Deng, date: 11/20/2016, view: 2115, Q&A: 0
Patient-derived xenograft (PDX) models for cancer research have recently attracted considerable attention in both the academy and industry (Hidalgo et al., 2014; Wilding and Bodmer, 2014). PDX models have been developed from different tumor types including lung cancer to improve the drug development process. These models are used for ...
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Determining the Influence of Small Molecules on Hypoxic Prostate Cancer Cell (DU-145) Viability Using Automated Cell Counting and a Cell Harvesting Protocol Authors:  John P Phelan, F Jerry Reen and Fergal O’Gara, date: 11/20/2016, view: 1282, Q&A: 0
Cell viability assays are an essential aspect of most cancer studies, however they usually require a considerable labor and time input. Here, instead of using the conventional microscopy and hemocytometer cell counting approach, we developed a cell harvesting protocol and combined it with the automated Countess Automated Cell Counter to generate ...
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Total Histone Acid Extraction of Colon Cancer HCT116 Cells Authors:  Lin-Lin Cao and Wei-Guo Zhu, date: 11/20/2016, view: 1342, Q&A: 0
Histone acid extraction assay is a popular method to determine histone modification levels in mammalian cells. It includes three steps: first, histones are released from chromatin by sulfuric acid; trichloroacetate (TCA) is then added to precipitate histones; and finally, histones are dissolved in double-distilled H2O (ddH2 ...
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Cell Biology

Quantitative Measurements of HIV-1 and Dextran Capture by Human Monocyte-derived Dendritic Cells (MDDCs) Authors:  Mickaël M. Ménager and Dan R. Littman, date: 11/20/2016, view: 1668, Q&A: 0
The aim of this protocol is to describe how to measure and quantify the amount of HIV-1 particles and dextran molecules internalized in human monocyte derived dendritic cells (MDDCs), using three different techniques: flow cytometry, quantitative PCR and confocal microscopy.
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Immunology

Assay to Evaluate BAL Fluid Regulation of Fibroblast α-SMA Expression Authors:  Jennifer L. Larson-Casey and A. Brent Carter, date: 11/20/2016, view: 1612, Q&A: 0
Because transforming growth factor-β (TGF-β1) induces differentiation of fibroblasts to myofibroblasts, we developed a protocol to evaluate alveolar macrophage-derived TGF-β1 regulation of lung fibroblast differentiation (Larson-Casey et al., 2016). The protocol evaluates the ability of mouse bronchoalveolar lavage (BAL) fluid to alter ...
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Determination of H2O2 Generation by pHPA Assay Authors:  Jennifer L. Larson-Casey and A. Brent Carter, date: 11/20/2016, view: 1640, Q&A: 0
The production of reactive oxygen species, including H2O2, is a process that can be used in signaling, cell death, or immune response. To quantify oxidative stress in cells, a fluorescence technique has been modified from a previously described method to measure H2O2 release from cells (Panus et al ...
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Isolation of Highly Pure Primary Mouse Alveolar Epithelial Type II Cells by Flow Cytometric Cell Sorting Authors:  Meenal Sinha and Clifford A. Lowell, date: 11/20/2016, view: 2681, Q&A: 0
In this protocol, we describe the method for isolating highly pure primary alveolar epithelial type II (ATII) cells from lungs of naïve mice. The method combines negative selection for a variety of lineage markers along with positive selection for EpCAM, a pan-epithelial cell marker. This method yields 2-3 x 106 ATII cells per mouse ...
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Analysis of Phagosomal Antigen Degradation by Flow Organellocytometry Authors:  Eik Hoffmann, Anne-Marie Pauwels, Andrés Alloatti, Fiorella Kotsias and Sebastian Amigorena, date: 11/20/2016, view: 1746, Q&A: 0
Professional phagocytes internalize self and non-self particles by phagocytosis to initiate innate immune responses. After internalization, the formed phagosome matures through fusion and fission events with endosomes and lysosomes to obtain a more acidic, oxidative and hydrolytic environment for the degradation of its cargo. Interestingly, ...
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Evaluation of Cross-presentation in Bone Marrow-derived Dendritic Cells in vitro and Splenic Dendritic Cells ex vivo Using Antigen-coated Beads Authors:  Andrés Alloatti, Fiorella Kotsias, Eik Hoffmann and Sebastian Amigorena, date: 11/20/2016, view: 1882, Q&A: 0
Antigen presentation by MHC class I molecules, also referred to as cross-presentation, elicits cytotoxic immune responses. In particular, dendritic cells (DC) are the most proficient cross-presenting cells, since they have developed unique means to control phagocytic and degradative pathways.
   This protocol allows the evaluation ...
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Microbiology

Heterologous Expression and Purification of the Magnesium Transporter A (MgtA) in Escherichia coli Authors:  Saranya Subramani and Jens Preben Morth, date: 11/20/2016, view: 1835, Q&A: 0
The magnesium transporter A (MgtA) is a magnesium transporting P-type ATPase present in prokaryotes and plants (Subramani et al., 2016). In Salmonella typhimurium and Escherichia coli (E. coli), MgtA is expressed only in magnesium limiting conditions and plays an important role in Mg2+ homeostasis ...
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Transfer of Large Contiguous DNA Fragments onto a Low Copy Plasmid or into the Bacterial Chromosome Authors:  Analise Z Reeves and Cammie F Lesser, date: 11/20/2016, view: 2072, Q&A: 0
Bacterial pathogenicity islands and other contiguous operons can be difficult to clone using conventional methods due to their large size. Here we describe a robust 3-step method to transfer large defined fragments of DNA from virulence plasmids or cosmids onto smaller autonomously replicating plasmids or directly into defined sites in the ...
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Cell-to-cell DNA Transfer among Thermus Species Authors:  Alba Blesa and José Berenguer, date: 11/20/2016, view: 1515, Q&A: 0
The ability to transfer DNA via direct cell-to-cell contact-dependent process similar to conjugation has been described in Thermus thermophilus (Tth). Here, we detail the mating experiment protocol involving the lateral transfer of thermostable antibiotic resistance markers (i.e., kanamycin: KmR; hygromycin: HygR ...
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Transformation of Thermus Species by Natural Competence Authors:  Alba Blesa and José Berenguer, date: 11/20/2016, view: 1406, Q&A: 0
Many Thermus species harbour genomes scourged with horizontally transferred signatures. Thermus thermophilus (Tth) has been characterized as naturally competent. The transformation protocol described here is based on the maximum DNA uptake rates registered at mid-exponential phase (OD600 0.3-0.4). Here we ...
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Biochemical Analysis of Caspase-8-dependent Proteolysis of IRF3 in Virus-infected Cells Authors:  Gayatri Subramanian, Karen Pan, Ritu Chakravarti and Saurabh Chattopadhyay, date: 11/20/2016, view: 1695, Q&A: 0
Interferon regulatory factor 3 (IRF3) is a transcription factor, which is critical for the antiviral response against a wide range of viruses (Hiscott, 2007; Ikushima et al., 2013). It gets activated in virus-infected cells via Toll like receptors (TLRs), RIG-I (retinoic acid inducible gene 1) like receptors (RLRs), cyclic GMP-AMP ...
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Halo Assay for Toxic Peptides and Other Compounds in Microorganisms Authors:  Houjian Cai, Melinda Hauser, Fred Naider and Jeffrey M. Becker, date: 11/20/2016, view: 1567, Q&A: 0
We describe an assay for determination of toxicity in S. cerevisiae involving spotting of a toxic peptide on a lawn of yeast cells. This assay may be generalized to determine toxicity of a variety of compounds by substituting a putative toxic compound in place of the peptide. The general protocol may also be used to determine toxicity of ...
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Uptake Assay for Radiolabeled Peptides in Yeast Authors:  Melinda Hauser, Houjian Cai, Fred Naider and Jeffrey M. Becker, date: 11/20/2016, view: 1547, Q&A: 0
We describe an assay for measuring the uptake of radioactive peptides into the yeast Saccharomyces cerevisiae. The methods presented here can be adapted to measure a variety of substrates transported into any bacterial or fungal cell via specific carrier-mediated systems.
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Neuroscience

In vitro Brainstem-spinal Cord Preparation from Newborn Rat Authors:  Jean-Patrick Le Gal, Angelo Nicolosi, Laurent Juvin and Didier Morin, date: 11/20/2016, view: 1566, Q&A: 0
The brainstem-spinal cord preparation of newborn rat contains neural networks able to produce motor output in absence of sensory feedback. These neural structures, commonly called central pattern generators (CPGs), are involved in many vital functions such as respiration (Morin and Viala, 2002; Giraudin et al., 2008) or locomotion (Juvin ...
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An in vitro Model of Neuron-macrophage Interaction to Generate Macrophages with Neurite Outgrowth Properties Authors:  Hyeok Jun Yun and Byung S. Kim, date: 11/20/2016, view: 1586, Q&A: 0
Macrophages are known to play beneficial roles in axon regeneration after nerve injury. To develop an in vitro model in which injury signals can elicit pro-regenerative macrophage activation, we established co-cultures consisting of adult dorsal root ganglia sensory neurons and peritoneal macrophages and added cAMP analogue dibutyryl ...
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Microinjection of Virus into Lumbar Enlargement of Spinal Dorsal Horn in Mice Authors:  Zhi-Jun Zhang, Peng-Bo Jing and Yong-Jing Gao, date: 11/20/2016, view: 2003, Q&A: 0
In order to explore the role of a specific gene/protein in the specific segment of spinal cord, the technique of intraspinal injection is particularly used to deliver viral vectors targeting the specific gene/protein. These viral vectors can knockdown or overexpress the specific gene/protein in specific cells (glial cells or neurons). In this ...
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Plant Science

Arabidopsis Seed Germination Assay with Gibberellic Acid Authors:  Chunmei Zhong, Hao Xu, Siting Ye, Shengchun Zhang and Xiaojing Wang, date: 11/20/2016, view: 2160, Q&A: 0
This assay analyzes Arabidopsis seed germination in response to gibberellic acid (GA). During seed imbibition, visible physiological changes allow precise determination of germination rate. This protocol utilizes a stereoscopic microscope to improve characterization of seed germination process.
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Documentation of floral secretory glands in Pleurothallidinae (Orchidaceae) using Scanning Electron Microscopy (SEM) Authors:  Adam P. Karremans, Bertie Joan van Heuven, Rob Langelaan and Barbara Gravendeel, date: 11/20/2016, view: 1597, Q&A: 0
A clear, step by step description of the treatment of orchid flowers, subtribe Pleurothallidinae, with Critical Point Drying for SEM is presented. It shows that a simple, short fixation and dehydration method prior to Critical Point Drying is sufficient to obtain good results.
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Visualising Differential Growth of Arabidopsis Epidermal Pavement Cells Using Thin Plate Spline Analysis Authors:  William Jonathan Armour, Deborah Anne Barton and Robyn Lynette Overall, date: 11/20/2016, view: 1600, Q&A: 0
Epidermal pavement cells in Arabidopsis leaves and cotyledons develop from relatively simple shapes to form complex cells that have multiple undulations of varying sizes. Analyzing the growth of individual parts of the cell wall boundaries over time is essential to understanding how pavement cells develop their complex shapes. Thin plate ...
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Cryopreservation Protocol for Chlamydomonas reinhardtii Authors:  Duanpeng Yang and Weiqi Li, date: 11/20/2016, view: 1620, Q&A: 0
Cryopreservation is commonly used for storing viable cells, tissues, organs or organisms at ultralow temperatures, and usually involves immersion in liquid nitrogen at -196 °C. Here we provide a detailed cryopreservation protocol for C. reinhardtii based on Crutchfield’s work (Crutchfield et al., 1999), with minor changes (Yang ...
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Stem Cell

Isolation and Culture of Human Adipose-derived Stem Cells from Subcutaneous and Visceral White Adipose Tissue Compartments Authors:  Xiaojia Ge, Shi Chi Leow, Durgalakshmi Sathiakumar, Walter Stünkel, Asim Shabbir, Jimmy Bok Yan So, Davide Lomanto and Craig McFarlane, date: 11/20/2016, view: 2048, Q&A: 0
Human Adipose-derived Stem/Stromal Cells (ASCs) have been widely used in stem cell and obesity research, as well as clinical applications including cell-based therapies, tissue engineering and reconstruction. Compared with mesenchymal stem cells (MSCs) derived from other tissues such as umbilical cord and bone marrow, isolation of ASCs from human ...
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Isolation and Primary Culture of Various Cell Types from Whole Human Endometrial Biopsies Authors:  Flavio Santos Vasconcelos Barros, Jan Joris Brosens and Paul John Brighton, date: 11/20/2016, view: 2053, Q&A: 0
The isolation and primary culture of cells from human endometrial biopsies provides valuable experimental material for reproductive and gynaecological research. Whole endometrial biopsies are collected from consenting women and digested with collagenase and DNase I to dissociate cells from the extracellular matrix. Cell populations are then ...
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