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Volume:4  Issue:12

June 20, 2014

Cancer Biology

Lysosomal Stability Assay

Authors: Nikolaj H. T. Petersen
Nikolaj H. T. PetersenAffiliation: Orphazyme ApS, Orphazyme ApS, Copenhagen, Denmark
For correspondence: nhtp@orphazyme.com
Bio-protocol author page: a1442
Thomas Kirkegaard
Thomas KirkegaardAffiliation: Orphazyme ApS, Orphazyme ApS, Copenhagen, Denmark
Bio-protocol author page: a1443
 and Marja Jäättelä
Marja JäätteläAffiliation: Cell Death and Metabolism Department, Danish Cancer Society Research Center, Copenhagen, Denmark
Bio-protocol author page: a1444
date: 6/20/2014, 4999 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1162 

[Abstract] This assay makes use of the dye Acridine Orange (AO) to determine the stability of lysosomes in living cells upon exposure to a confocal microscope laser.


Figure 1. Acridine Orange

AO is a lipophilic amine that readily diffuses into cells (Figure 1). ...




Article identifier: bio-protocol.org/e1162

Cell Biology

EML Erythroid and Neutrophil Differentiation Protocols

Authors: Cristina Pina
Cristina PinaAffiliation: Department of Haematology, University of Cambridge, Cambridge, UK
For correspondence: cp533@medschl.cam.ac.uk
Bio-protocol author page: a1418
Cristina Fugazza
Cristina FugazzaAffiliation: Dipartimento de Biotecnologie e Bioscienze, Universita degli Studi de Milano-Bicocca, Milan, Italy
Bio-protocol author page: a1419
 and Tariq Enver
Tariq EnverAffiliation: UCL Cancer Institute, University College, London, UK
Bio-protocol author page: a1420
date: 6/20/2014, 3814 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1151 

[Abstract] Erythroid-Myeloid-Lymphoid cells (EML) are a multipotent haematopoietic cell line of mouse bone marrow origin capable of long-term maintenance in vitro in the presence of SCF (stem cell factor) (Tsai et al., 1994). The self-renewal capacity of the EML ...

Article identifier: bio-protocol.org/e1151

Immunology

Bone Marrow Derived Eosinophil Cultures

Authors: Thomas X. Lu
Thomas X. LuAffiliation: Division of Pediatrics, Cincinnati Children's Hospital Medical Center / University of Cincinnati, Cincinnati, USA
Bio-protocol author page: a1440
 and Marc E. Rothenberg
Marc E. RothenbergAffiliation: Division of Pediatrics, Cincinnati Children's Hospital Medical Center / University of Cincinnati, Cincinnati, USA
For correspondence: marc.rothenberg@cchmc.org
Bio-protocol author page: a1441
date: 6/20/2014, 3730 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1161 

[Abstract] Eosinophils are multifunctional effector cells implicated in the pathogenesis of a variety of diseases including asthma, eosinophil gastrointestinal disorders and helminth infection. Mouse bone marrow derived progenitor cells can be differentiated into ...

Article identifier: bio-protocol.org/e1161

Microbiology

A SYBR Green-based Real Time RT-PCR Assay for Detection of the Emerging H7N9 Virus

Authors: Zheng Zhu
Zheng ZhuAffiliation: Institute of Pathogenic Microbiology, Jiangsu Provincial Center for Disease Control and Prevention, Nanjing, China
For correspondence: zhengzhu@jscdc.cn
Bio-protocol author page: a1416
 and Lunbiao Cui
Lunbiao CuiAffiliation: Institute of Pathogenic Microbiology, Jiangsu Provincial Center for Disease Control and Prevention, Nanjing, China
Bio-protocol author page: a1417
date: 6/20/2014, 2650 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1150 

[Abstract] Most recently a novel avian-origin influenza A (H7N9) virus emerged in China and has been associated with lots of human infection and fatal cases. Molecular diagnostic methods are thus urgently needed in public health laboratories. We developed a SYBR ...

Article identifier: bio-protocol.org/e1150

Determination of Oxygen Respiration Rates in Wetted Developmentally Arrested Spores of Streptomyces Species

Authors: Marco Fischer
Marco FischerAffiliation: Department of Microbiology, Martin-Luther University Halle-Wittenberg, Halle (Saale), Germany
Bio-protocol author page: a1422
Dörte Falke
Dörte FalkeAffiliation: Department of Microbiology, Martin-Luther University Halle-Wittenberg, Halle (Saale), Germany
Bio-protocol author page: a1423
 and R. Gary Sawers
R. Gary SawersAffiliation: Department of Microbiology, Martin-Luther University Halle-Wittenberg, Halle (Saale), Germany
For correspondence: gary.sawers@mikrobiologie.uni-halle.de
Bio-protocol author page: a1424
date: 6/20/2014, 2959 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1153 

[Abstract] Streptomyces species produce spores, which, while not as robust as endospores of Bacillus or Clostridium species, are capable of surviving for months or even years (Hopwood, 2006). During this time these spores remain viable, surviving by slowly degrading ...

Article identifier: bio-protocol.org/e1153

Rapid Nitrate Reduction Assay with Intact Microbial Cells or Spores

Authors: Marco Fischer
Marco FischerAffiliation: Department of Microbiology, Martin-Luther University Halle-Wittenberg, Halle (Saale), Germany
Bio-protocol author page: a1422
Dörte Falke
Dörte FalkeAffiliation: Department of Microbiology, Martin-Luther University Halle-Wittenberg, Halle (Saale), Germany
Bio-protocol author page: a1423
 and R. Gary Sawers
R. Gary SawersAffiliation: Department of Microbiology, Martin-Luther University Halle-Wittenberg, Halle (Saale), Germany
For correspondence: gary.sawers@mikrobiologie.uni-halle.de
Bio-protocol author page: a1424
date: 6/20/2014, 2847 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1154 

[Abstract] Many microorganisms have the capacity to use nitrate as a respiratory electron acceptor. Reduction of nitrate is catalyzed by a multi-subunit nitrate reductase that is often located associated with the cytoplasmic membrane and has its active site oriented ...

Article identifier: bio-protocol.org/e1154

Analysis of Mycobacterial Protein Secretion

Authors: Alka Mehra
Alka MehraAffiliation: Department of Medicine, Division of Infectious Diseases, New York University School of Medicine, New York, USA
For correspondence: alkam08@gmail.com
Bio-protocol author page: a1435
 and  Jennifer A. Philips
Jennifer A. PhilipsAffiliation: Department of Medicine, Division of Infectious Diseases, New York University School of Medicine, New York, USA
For correspondence: jphilips@dom.wustl.edu
Bio-protocol author page: a1436
date: 6/20/2014, 4317 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1159 

[Abstract] Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis. Analysis of proteins secreted by Mtb has been of interest to the field of tuberculosis research since certain secreted proteins interact with the host to promote virulence, while ...

Article identifier: bio-protocol.org/e1159

Plant Science

Protoplast Preparation and Determination of Cell Death

Author: Antoine Danon
Antoine DanonAffiliation: UMR 8226, Laboratoire de Biologie Moléculaire et Cellulaire des Eucaryotes, Sorbonne Universités, UPMC Univ Paris 06, Centre National de la Recherche Scientifique, Institut de Biologie Physico-Chimique, Paris, France
For correspondence: antoine.danon@ibpc.fr
Bio-protocol author page: a1415
date: 6/20/2014, 4423 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1149 

[Abstract] The protoplasts assay constitutes a powerful tool that allows an easy uptake of active agents and a precise quantification of cell death induction in different populations. Our study showed that the basal level of cell death in our controls is low and ...

Article identifier: bio-protocol.org/e1149

MTV1 Pull-down Assay in Arabidopsis

Author: Michael Sauer
Michael SauerAffiliation: Institute of Biochemistry and Biology, Plant Physiology, University of Potsdam, Potsdam, Germany
For correspondence: michael.sauer@uni-potsdam.de
Bio-protocol author page: a1421
date: 6/20/2014, 3506 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1152 

[Abstract] This protocol is an example of how to analyse suspected interactions between proteins using a pull-down assay (Sauer et al., 2013). A bait protein of interest (in this case, MTV1 of Arabidopsis thaliana) is fused to a GST tag and expressed in bacteria. ...

Article identifier: bio-protocol.org/e1152

Deflagellation and Regeneration in Chlamydomonas

Author: William Dentler
William DentlerAffiliation: Department of Molecular Biosciences, University of Kansas, Lawrence, USA
For correspondence: wdent@ku.edu
Bio-protocol author page: a551
date: 6/20/2014, 4291 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1155 

[Abstract] Eukaryotic cilia/flagella are one of the only cellular structures that can be removed without injuring cells, can be highly purified for biochemical analysis, and, in many cells, can be completely reassembled within 90 minutes. Following amputation, ...

Article identifier: bio-protocol.org/e1155

Ciliary and Flagellar Membrane Vesicle (Ectosome) Purification

Author: William Dentler
William DentlerAffiliation: Department of Molecular Biosciences, University of Kansas, Lawrence, USA
For correspondence: wdent@ku.edu
Bio-protocol author page: a551
date: 6/20/2014, 3369 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1156 

[Abstract] Eukaryotic cilia/flagella are ideal organelles for the analysis of membrane trafficking, membrane assembly, and the functions of a variety of signal transduction molecules. Cilia are peninsular organelles and the membrane lipids, membrane proteins, and ...

Article identifier: bio-protocol.org/e1156

In organello Protein Synthesis

Authors: Malgorzata Kwasniak-Owczarek
Malgorzata Kwasniak-Owczarek Affiliation: Department of Biotechnology, University of Wroclaw, Wroclaw, Poland
Bio-protocol author page: a1425
 and Hanna Janska
Hanna JanskaAffiliation: Department of Biotechnology, University of Wroclaw, Wroclaw, Poland
For correspondence: janska@ibmb.uni.wroc.pl
Bio-protocol author page: a1426
date: 6/20/2014, 3519 views, 1 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1157 

[Abstract] In organello protein synthesis method allows the analysis of mitochondrial translation products. The principle of this method relies on incubation of isolated intact mitochondria with radiolabeled amino acids such as 35S methionine. After protein synthesis, ...

Article identifier: bio-protocol.org/e1157

Determination of Luciferase Activity in Arabidopsis seedling

Authors: Mohan TC
Mohan TCAffiliation: Department of Plant Molecular Genetics, Centro Nacional de Biotecnología, Madrid, Spain
Bio-protocol author page: a1437
Gabriel Castrillo
Gabriel CastrilloAffiliation: Department of Plant Molecular Genetics, Centro Nacional de Biotecnología, Madrid, Spain
Bio-protocol author page: a1438
 and Antonio Leyva
Antonio LeyvaAffiliation: Department of Plant Molecular Genetics, Centro Nacional de Biotecnología, Madrid, Spain
For correspondence: aleyva@cnb.csic.es
Bio-protocol author page: a1439
date: 6/20/2014, 3698 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1160 

[Abstract] There are different direct and indirect methods available to study gene expression in plant systems. In this protocol we describe a modified expression assay using transgenic plants expressing the luciferase gene under the control of phosphate transporter ...

Article identifier: bio-protocol.org/e1160

Systems Biology

ChIP-Seq in Candida albicans

Authors: Sadri Znaidi
Sadri ZnaidiAffiliation: Genomes and Genetics Department, Institut Pasteur, Paris, France
For correspondence: sadri.znaidi@pasteur.fr
Bio-protocol author page: a1427
Caroline Proux
Caroline ProuxAffiliation: Genomes and Genetics Department, Institut Pasteur, Paris, France
Bio-protocol author page: a1428
Sandra Weber
Sandra WeberAffiliation: Biochemistry Department, Institute for Research in Immunology and Cancer and University of Montreal, Montreal, Canada
Bio-protocol author page: a1429
Simon Drouin
Simon DrouinAffiliation: Department of Medicine, Clinical Research Institute of Montreal and University of Montreal, Montreal, Canada
Bio-protocol author page: a1430
François Robert
François RobertAffiliation: Department of Medicine, Clinical Research Institute of Montreal and University of Montreal, Montreal, Canada
Bio-protocol author page: a1431
Martine Raymond
Martine RaymondAffiliation: Biochemistry Department, Institute for Research in Immunology and Cancer and University of Montreal, Montreal, Canada
Bio-protocol author page: a1432
Jean-Yves Coppée
Jean-Yves CoppéeAffiliation: Genomes and Genetics Department, Institut Pasteur, Paris, France
Bio-protocol author page: a1433
 and Christophe d’Enfert
Christophe d’EnfertAffiliation: Genomes and Genetics Department, Institut Pasteur, Paris, France
For correspondence: christophe.denfert@pasteur.fr
Bio-protocol author page: a1434
date: 6/20/2014, 5654 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1158 

[Abstract] Systems biology approaches can be used to study the regulatory interactions occurring between many components of the biological system at the whole-genome level and decipher the circuitries implicated in the regulation of cellular processes, including ...

Article identifier: bio-protocol.org/e1158