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Volume:5  Issue:11

June 05, 2015

Cell Biology

Dictyostelium Cultivation, Transfection, Microscopy and Fractionation

Authors: Jennifer Hirst
Jennifer HirstAffiliation: Cambridge Institute for Medical Research, University of Cambridge, Cambridge, UK
For correspondence: jh228@cam.ac.uk
Bio-protocol author page: a2250
Robert R Kay
Robert R KayAffiliation: MRC Laboratory of Molecular Biology, Cambridge, UK
Bio-protocol author page: a2251
 and David Traynor
David TraynorAffiliation: MRC Laboratory of Molecular Biology, Cambridge, UK
For correspondence: dt101@mrc-lmb.cam.ac.uk
Bio-protocol author page: a2252
date: 6/5/2015, 3337 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1485 

[Abstract] The real time visualisation of fluorescently tagged proteins in live cells using ever more sophisticated microscopes has greatly increased our understanding of the dynamics of key proteins during fundamental physiological processes such as cell locomotion, ...

Article identifier: bio-protocol.org/e1485

FRAP Analysis of LET-23::GFP in the Vulval Epithelial Cells of Living Caenorhabditis elegans Larvae

Authors: Michael Walser
Michael WalserAffiliation: University of Zurich, Institute of Molecular Life Sciences, Winterthurerstrasse, Zurich
Bio-protocol author page: a2260
Alex Hajnal
Alex HajnalAffiliation: University of Zurich, Institute of Molecular Life Sciences, Winterthurerstrasse, Zurich
Bio-protocol author page: a2261
 and Juan M. Escobar-Restrepo
Juan M. Escobar-RestrepoAffiliation: University of Zurich, Institute of Molecular Life Sciences, Winterthurerstrasse, Zurich
For correspondence: juan.escobar@imls.uzh.ch
Bio-protocol author page: a2262
date: 6/5/2015, 1948 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1489 

[Abstract] The Caenorhabditis elegans (C. elegans) vulva is a well-established system to study organ development as the molecular mechanisms that govern its formation are conserved in animals. Of special interest is the EGFR/RAS/MAPK signaling pathway that is required ...

Article identifier: bio-protocol.org/e1489

Immunology

Extraction and Identification of T Cell Stimulatory Self-lipid Antigens

Authors: Marco Lepore
Marco LeporeAffiliation: Experimental Immunology, Department of Biomedicine, University Hospital Basel, Basel, Switzerland
Bio-protocol author page: a2266
Sebastiano Sansano
Sebastiano SansanoAffiliation: Experimental Immunology, Department of Biomedicine, University Hospital Basel, Basel, Switzerland
Bio-protocol author page: a2267
Claudia de Lalla
Claudia de LallaAffiliation: Experimental Immunology Unit, Division of Immunology, Transplantation and Infectious Diseases, San Raffaele Scientific Institute, Milano, Italy
Bio-protocol author page: a2268
Paolo Dellabona
Paolo DellabonaAffiliation: Experimental Immunology Unit, Division of Immunology, Transplantation and Infectious Diseases, San Raffaele Scientific Institute, Milano, Italy
Bio-protocol author page: a2269
Giulia Casorati
Giulia CasoratiAffiliation: Experimental Immunology Unit, Division of Immunology, Transplantation and Infectious Diseases, San Raffaele Scientific Institute, Milano, Italy
Bio-protocol author page: a2270
Gennaro De Libero
Gennaro De LiberoAffiliation 1: Experimental Immunology, Department of Biomedicine, University Hospital Basel, Basel, Switzerland
Affiliation 2: Singapore Immunology Network (SIgN), Agency for Science, Technology, and Research, Singapore
Bio-protocol author page: a2271
 and Lucia Mori
Lucia MoriAffiliation 1: Experimental Immunology, Department of Biomedicine, University Hospital Basel, Basel, Switzerland
Affiliation 2: Singapore Immunology Network (SIgN), Agency for Science, Technology, and Research, Singapore
For correspondence: lucia.mori@unibas.ch
Bio-protocol author page: a2272
date: 6/5/2015, 2128 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1491 

[Abstract] Autoreactive T cells restricted to CD1 molecules and specific for endogenous lipids are abundant in human blood (de Jong et al., 2010; de Lalla et al., 2011). A few self-lipid molecules recognized by diverse individual T cell clones and accumulated within ...

Article identifier: bio-protocol.org/e1491

Cytokine-Stimulated Phosphoflow of Whole Blood Using CyTOF Mass Cytometry

Authors: Rosemary Fernandez
Rosemary FernandezAffiliation: Human Immune Monitoring Center, Stanford University, Stanford, USA
Bio-protocol author page: a2282
 and Holden Maecker
Holden MaeckerAffiliation: Human Immune Monitoring Core, Institute of Immunity, Transplantation & Infection, Stanford University School of Medicine, Stanford, USA
For correspondence: maecker@stanford.edu
Bio-protocol author page: a1861
date: 6/5/2015, 3270 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1495 

[Abstract] The ability to assess the function of a range of cytokine, antigen receptor, and Toll-like receptor (TLR) signaling pathways in a range of immune cells could provide a kind of fingerprint of the state of the human immune system. The mass cytometry or ...

Article identifier: bio-protocol.org/e1495

Cytokine-stimulated Phosphoflow of PBMC Using CyTOF Mass Cytometry

Authors: Rosemary Fernandez
Rosemary FernandezAffiliation: Human Immune Monitoring Center, Stanford University, Stanford, USA
Bio-protocol author page: a2282
 and Holden Maecker
Holden MaeckerAffiliation: Human Immune Monitoring Center, Stanford University , Stanford, USA
For correspondence: maecker@stanford.edu
Bio-protocol author page: a1861
date: 6/5/2015, 3844 views, 1 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1496 

[Abstract] Phosphorylation of tyrosine, serine, and threonine residues is critical for the control of protein activity involved in various cellular events. An assortment of kinases and phosphatases regulate intracellular protein phosphorylation in many different ...

Article identifier: bio-protocol.org/e1496

Microbiology

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Quantification of Total and 2-LTR (Long terminal repeat) HIV DNA, HIV RNA and Herpesvirus DNA in PBMCs

Authors: Marta Massanella
Marta MassanellaAffiliation: University of California San Diego, La Jolla, USA
For correspondence: mmassanellaluna@ucsd.edu
Bio-protocol author page: a2185
Sara Gianella
Sara GianellaAffiliation: University of California San Diego, La Jolla, USA
Bio-protocol author page: a2187
Steven M. Lada
Steven M. LadaAffiliation: University of California San Diego, La Jolla, USA
Bio-protocol author page: a2274
Douglas D. Richman
Douglas D. RichmanAffiliation 1: University of California San Diego, La Jolla, USA
Affiliation 2: Veterans Affairs San Diego Healthcare System, La Jolla, USA
Bio-protocol author page: a2275
 and Matthew C. Strain
Matthew C. StrainAffiliation: University of California San Diego, La Jolla, USA
For correspondence: mstrain@ucsd.edu
Bio-protocol author page: a2276
date: 6/5/2015, 3131 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1492 

[Abstract] Almost all individuals infected with human immunodeficiency virus (HIV) are also infected with cytomegalovirus (CMV) and Epstein Barr virus (EBV). The aims of our studies have included characterizing and measuring the latent HIV reservoir and understanding ...

Article identifier: bio-protocol.org/e1492

Molecular Biology

DNA Damage Sensitivity Assays in Caenorhabditis elegans

Authors: Hyun-Min Kim
Hyun-Min KimAffiliation: Department of Genetics, Harvard Medical School, Boston, USA
For correspondence: hkim@genetics.med.harvard.edu
Bio-protocol author page: a2256
 and Monica P. Colaiácovo
Monica P. ColaiácovoAffiliation: Department of Genetics, Harvard Medical School, Boston, USA
For correspondence: mcolaiacovo@genetics.med.harvard.edu
Bio-protocol author page: a2257
date: 6/5/2015, 2822 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1487 

[Abstract] C. elegans has served as a genetically tractable multicellular model system to examine DNA damage-induced genotoxic stress which threatens genome integrity. Importantly, the high degree of conservation shared between worms and humans offers the advantage ...

Article identifier: bio-protocol.org/e1487

Enzymatic Activity Assays for Base Excision Repair Enzymes in Cell Extracts from Vertebrate Cells

Authors: Melike Çağlayan
Melike ÇağlayanAffiliation: Genome Integrity and Structural Biology Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina, USA
Bio-protocol author page: a1849
Julie K. Horton
Julie K. HortonAffiliation: Genome Integrity and Structural Biology Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina, USA
Bio-protocol author page: a2278
 and Samuel H. Wilson
Samuel H. WilsonAffiliation: Genome Integrity and Structural Biology Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina, USA
For correspondence: wilson5@niehs.nih.gov
Bio-protocol author page: a1850
date: 6/5/2015, 1816 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1493 

[Abstract] We previously reported enzymatic activity assays for the base excision repair (BER) enzymes DNA polymerase β (pol β), aprataxin (APTX), and flap endonuclease 1 (FEN1) in cell extracts from Saccharomyces cerevisiae (Çağlayan and Wilson, 2014). Here, we ...

Article identifier: bio-protocol.org/e1493

Plant Science

Determination of the Developmental Origin of Seeds Containing Endosperm Using Flow Cytometric Analysis

Authors: Christian Sailer
Christian SailerAffiliation: Institute of Plant Biology & Zürich-Basel Plant Science Center, University of Zürich, Zürich, Switzerland
For correspondence: christian.sailer@access.uzh.ch
Bio-protocol author page: a2246
Anja Schmidt
Anja SchmidtAffiliation: Institute of Plant Biology & Zürich-Basel Plant Science Center, University of Zürich, Zürich, Switzerland
Bio-protocol author page: a2247
 and Ueli Grossniklaus
Ueli GrossniklausAffiliation: Institute of Plant Biology & Zürich-Basel Plant Science Center, University of Zürich, Zürich, Switzerland
For correspondence: grossnik@botinst.uzh.ch
Bio-protocol author page: a2248
date: 6/5/2015, 2080 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1484 

[Abstract] Seeds derived from a diploid, sexual plant typically contain a 2n embryo (n+n) and 3n endosperm, a ratio characteristic for most flowering plants. However, this ratio is altered in apomictic species, which reproduce asexually through seeds (Koltunow ...

Article identifier: bio-protocol.org/e1484

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Magnaporthe oryzae Inoculation of Rice Seedlings by Spraying with a Spore Suspension

Authors: Aya Akagi
Aya AkagiAffiliation: Disease Resistant Crops Research Unit, National Institute of Agrobiological Sciences, Tsukuba, Japan
Present address: Bayer CropScience, Tokyo, Japan
Bio-protocol author page: a2253
Chang-Jie Jiang
Chang-Jie JiangAffiliation: Disease Resistant Crops Research Unit, National Institute of Agrobiological Sciences, Tsukuba, Japan
Bio-protocol author page: a2254
 and Hiroshi Takatsuji
Hiroshi TakatsujiAffiliation: Disease Resistant Crops Research Unit, National Institute of Agrobiological Sciences, Tsukuba, Japan
For correspondence: takatsuh@affrc.go.jp
Bio-protocol author page: a2255
date: 6/5/2015, 2821 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1486 

[Abstract] Fungal blast caused by Magnaporthe oryzae (M. oryzae) leads to a serious yield loss of rice. Appropriate assessment of disease occurrence is necessary to investigate the nature of the disease and plant strategies to resist the disease. We describe our ...

Article identifier: bio-protocol.org/e1486

Simple Digital Photography for Assessing Biomass and Leaf Area Index in Cereals

Authors: Jaume Casadesús
Jaume CasadesúsAffiliation: Institut de Recerca i Tecnologia Agroalimentàries, IRTA, Parc de Gardeny - Edifici Fruitcentre, Lleida, Spain
For correspondence: jaume.casadesus@irta.cat
Bio-protocol author page: a2258
 and Dolors Villegas
Dolors VillegasAffiliation: Institut de Recerca i Tecnologia Agroalimentàries, IRTA, Av. Rovira Roure, Lleida, Spain
For correspondence: dolors.villegas@irta.cat
Bio-protocol author page: a2259
date: 6/5/2015, 2689 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1488 

[Abstract] These instructions refer to obtaining fast and low-labour estimates of ground cover, leaf area index and green biomass for a large number of plots, as those encountered in cereal breeding programs. The procedure includes obtaining the pictures in the ...

Article identifier: bio-protocol.org/e1488

Measurement of Net High-affinity Urea Uptake in Maize Plants

Authors: Laura Zanin
Laura ZaninAffiliation: Dipartimento di Scienze Agrarie e Ambientali, University of Udine, Udine, Italy
For correspondence: laura.zanin@uniud.it
Bio-protocol author page: a2263
Nicola Tomasi
Nicola TomasiAffiliation: Dipartimento di Scienze Agrarie e Ambientali, University of Udine, Udine, Italy
Bio-protocol author page: a2264
 and Roberto Pinton
Roberto PintonAffiliation: Dipartimento di Scienze Agrarie e Ambientali, University of Udine, Udine, Italy
Bio-protocol author page: a2265
date: 6/5/2015, 1899 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1490 

[Abstract] Despite its extensive use as a nitrogen fertilizer, the role of urea as a directly accessible nitrogen source for crop plants is still poorly understood. So far, the physiological and molecular aspects of urea acquisition have been investigated only ...

Article identifier: bio-protocol.org/e1490

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Cell Wall Biomass Preparation and Fourier Transform Mid-infrared (FTIR) Spectroscopy to Study Cell Wall Composition

Authors: Ricardo M. F. da Costa
Ricardo M. F. da CostaAffiliation: Institute of Biological, Environmental and Rural Sciences, Aberystwyth University, Ceredigion, UK
For correspondence: dacosta.rmf@gmail.com
Bio-protocol author page: a2279
Gordon G. Allison
Gordon G. AllisonAffiliation: Institute of Biological, Environmental and Rural Sciences, Aberystwyth University, Ceredigion, UK
For correspondence: goa@aber.ac.uk
Bio-protocol author page: a2280
 and Maurice Bosch
Maurice BoschAffiliation: Institute of Biological, Environmental and Rural Sciences, Aberystwyth University, Ceredigion, UK
Bio-protocol author page: a2281
date: 6/5/2015, 2038 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1494 

[Abstract] Plant cell wall biomass is an abundant and renewable organic resource. Of the polymers it encloses, cellulose and hemicellulose are regarded as a raw material for the production of fuels and other products (Klemm et al., 2005; Slavov et al., 2013). Nonetheless, ...

Article identifier: bio-protocol.org/e1494

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Quantitative Image Analysis of Membrane Microdomains Labelled by Fluorescently Tagged Proteins in Arabidopsis thaliana and Nicotiana benthamiana

Authors: Iris K. Jarsch
Iris K. JarschAffiliation: Ludwig-Maximilians-Universität München, Planegg-Martinsried, Germany
Present address: Gurdon Institute, University of Cambridge, Cambridge, UK
Bio-protocol author page: a2283
 and Thomas Ott
Thomas OttAffiliation: Ludwig-Maximilians-Universität München, Planegg-Martinsried, Germany
For correspondence: Thomas.Ott@bio.lmu.de
Bio-protocol author page: a2284
date: 6/5/2015, 2075 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1497 

[Abstract] We have recently characterized co-existing membrane microdomains that are labeled by different proteins in living plant cells (Jarsch et al., 2014). For this approach we first created a digital fingerprint for each of the twenty marker proteins using ...

Article identifier: bio-protocol.org/e1497