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A peer-reviewed protocol journal. No publication fee; no access fee.
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Volume 8, 2018
Volume 7, 2017
Volume 6, 2016
Volume 5, 2015
Volume 4, 2014
Volume 3, 2013
Volume 2, 2012
Volume 1, 2011
Past Issues in 2016
Volume: 6 Issue: 23
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Cancer Biology
DNA Damage Induction by Laser Microirradiation
Authors:
Marianna Tampere
and
Oliver Mortusewicz
,
date:
12/05/2016,
view:
7233,
Q&A:
1
Genome instability can lead to cell death, senescence and cancerous transformation. Specific repair pathways have evolved to prevent accumulation of DNA lesions. Studying these highly dynamic and specific repair pathways requires precise spatial and temporal resolution, which can be achieved through a combination of laser microirradiaiton and live ...
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Lentiviral shRNA Screen to Identify Epithelial Integrity Regulating Genes in MCF10A 3D Culture
Authors:
Elsa Marques
and
Juha Klefström
,
date:
12/05/2016,
view:
4061,
Q&A:
0
MCF10A 3D culture system provides a reductionist model of glandular mammary epithelium which is widely used to study development of glandular architecture, the role of cell polarity and epithelial integrity in control of epithelial cell functions, and mechanisms of breast cancer. Here we describe how to use shRNA screening approach to identify ...
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Cell Biology
Lymphocyte Isolation, Th17 Cell Differentiation, Activation, and Staining
Authors:
Pawan Kumar
and
Jay K Kolls
,
date:
12/05/2016,
view:
4737,
Q&A:
0
In vitro
Th17 (α, β T helper cell which produce IL-17A, IL-17F and IL-22) differentiation has been routinely used for functional T cells studies. Here we describe a method for Th17 cell differentiation.
More >>
Analysis of Myosin II Minifilament Orientation at Epithelial Zonula Adherens
Authors:
Magdalene Michael
,
Xuan Liang
and
Guillermo A. Gomez
,
date:
12/05/2016,
view:
3381,
Q&A:
0
Non-muscle myosin II (NMII) form bipolar filaments, which bind F-actin to exert cellular contractility during physiological processes (Vicente-Manzanares
et al
., 2009). Using a combinatorial approach to fluorescently label both N- and C-termini of the NMII heavy chain, recent works have demonstrated the ability to visualize NMII bipolar ...
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Isolation of Latex Bead Phagosomes from
Dictyostelium
for
in vitro
Functional Assays
Authors:
Ashwin D’Souza
,
Paulomi Sanghavi
,
Ashim Rai
,
Divya Pathak
and
Roop Mallik
,
date:
12/05/2016,
view:
4157,
Q&A:
0
We describe a protocol to purify latex bead phagosomes (LBPs) from
Dictyostelium
cells. These can be later used for various
in vitro
functional assays. For instance, we use these LBPs to understand the microtubule motor-driven transport on
in vitro
polymerized microtubules. Phagosomes are allowed to mature for defined ...
More >>
Developmental Biology
Vascular Smooth Muscle Cell Isolation and Culture from Mouse Aorta
Authors:
Callie S. Kwartler
,
Ping Zhou
,
Shao-Qing Kuang
,
Xue-Yan Duan
,
Limin Gong
and
Dianna M. Milewicz
,
date:
12/05/2016,
view:
7161,
Q&A:
0
Vascular smooth muscle cells (SMC) in the ascending thoracic aorta arise from neural crest cells, whereas SMCs in the descending aorta are derived from the presomitic mesoderm. SMCs play important roles in cardiovascular development and aortic aneurysm formation. This protocol describes the detailed process for explanting ascending and descending ...
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Intracellular Assessment of ATP Levels in
Caenorhabditis elegans
Authors:
Konstantinos Palikaras
and
Nektarios Tavernarakis
,
date:
12/05/2016,
view:
4409,
Q&A:
0
Eukaryotic cells heavily depend on adenosine triphosphate (ATP) generated by oxidative phosphorylation (OXPHOS) within mitochondria. ATP is the major energy currency molecule, which fuels cell to carry out numerous processes, including growth, differentiation, transportation and cell death among others (Khakh and Burnstock, 2009). Therefore, ATP ...
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Measuring Oxygen Consumption Rate in
Caenorhabditis elegans
Authors:
Konstantinos Palikaras
and
Nektarios Tavernarakis
,
date:
12/05/2016,
view:
4391,
Q&A:
0
The rate of oxygen consumption is a vital marker indicating cellular function during lifetime under normal or metabolically challenged conditions. It is used broadly to study mitochondrial function (Artal-Sanz and Tavernarakis, 2009; Palikaras
et al
., 2015; Ryu
et al
., 2016) or investigate factors mediating the switch from ...
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Microbiology
Murine Leukemia Virus (MLV)-based Coronavirus Spike-pseudotyped Particle Production and Infection
Authors:
Jean Kaoru Millet
and
Gary R. Whittaker
,
date:
12/05/2016,
view:
3957,
Q&A:
0
Viral pseudotyped particles (pp) are enveloped virus particles, typically derived from retroviruses or rhabdoviruses, that harbor heterologous envelope glycoproteins on their surface and a genome lacking essential genes. These synthetic viral particles are safer surrogates of native viruses and acquire the tropism and host entry pathway ...
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In vitro
Autophosphorylation and Phosphotransfer Assay of Cyanobacterial Histidine Kinase 2
Author:
Iskander M. Ibrahim
,
date:
12/05/2016,
view:
4413,
Q&A:
0
This is a detailed protocol of an autophosphorylation and phosphotransfer activities of
Synechocystis
sp. PCC 6803 full-length Histidine Kinase 2 (Hik2) protein described by Ibrahim
et al
., 2016. In this protocol, radioactively labelled ATP was used to study an autophosphorylation and phosphotransfer activity of the full-length ...
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Preparation of Purified Gram-positive Bacterial Cell Wall and Detection in Placenta and Fetal Tissues
Authors:
Beth Mann
,
Lip Nam Loh
,
Geli Gao
and
Elaine Tuomanen
,
date:
12/05/2016,
view:
3976,
Q&A:
0
Cell wall is a complex biopolymer on the surface of all Gram-positive bacteria. During infection, cell wall is recognized by the innate immune receptor Toll-like receptor 2 causing intense inflammation and tissue damage. In animal models, cell wall traffics from the blood stream to many organs in the body, including brain, heart, placenta and ...
More >>
Single Cell Flow Cytometry Assay for Peptide Uptake by Bacteria
Authors:
Monica Benincasa
,
Quentin Barrière
,
Giulia Runti
,
Olivier Pierre
,
Mick Bourge
,
Marco Scocchi
and
Peter Mergaert
,
date:
12/05/2016,
view:
4697,
Q&A:
0
Antimicrobial peptides (AMPs) can target the bacterial envelope or alternatively have intracellular targets. The latter requires uptake of the peptide by the bacterial cells. The bacterial internalization of an AMP can be evaluated by a fluorescence-based method that couples the use of the fluorescently labelled AMP to the fluorescence quencher ...
More >>
Mouse Model of Dengue Virus Infection with Serotypes 1 and 2 Clinical Isolates
Authors:
Satoru Watanabe
,
Kitti Wing Ki Chan
and
Subhash G. Vasudevan
,
date:
12/05/2016,
view:
4272,
Q&A:
0
Dengue is a global public health threat caused by infection with any of the 4 related dengue virus serotypes (DENV1-4). Clinical manifestations range from self-limiting febrile illness, known as dengue fever (DF), to life-threatening severe diseases, such as dengue hemorrhagic fever (DHF) or dengue shock syndrome (DSS). Most cases of DHF/DSS are ...
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Measurement of Intracellular Calcium Concentration in
Pseudomonas aeruginosa
Authors:
Manita Guragain
,
Anthony K. Campbell
and
Marianna A. Patrauchan
,
date:
12/05/2016,
view:
3577,
Q&A:
0
Characterization of the molecular mechanisms of calcium (Ca
2+
) regulation of bacterial physiology and virulence requires tools enabling measuring and monitoring the intracellular levels of free calcium (Ca
2+
in
). Here, we describe a protocol optimized to use a recombinantly expressed Ca
2+
-binding protein, ...
More >>
Pyocyanin Extraction and Quantitative Analysis in Swarming
Pseudomonas aeruginosa
Authors:
Michelle M. King
,
Manita Guragain
,
Svetlana A. Sarkisova
and
Marianna A. Patrauchan
,
date:
12/05/2016,
view:
5885,
Q&A:
0
This protocol describes the quantification of pyocyanin extracted from swarming colonies of
Pseudomonas aeruginosa
. Pyocyanin is a secondary metabolite and a major virulence factor, whose production is inducible and varies highly under different growth conditions. The protocol is based on the earlier developed chloroform/HCl extraction of ...
More >>
Neuroscience
Various Modes of Spinal Cord Injury to Study Regeneration in Adult Zebrafish
Authors:
Subhra Prakash Hui
and
Sukla Ghosh
,
date:
12/05/2016,
view:
5271,
Q&A:
0
Spinal cord injury (SCI) in mammals leads to failure of both sensory and motor functions, due to lack of axonal regrowth below the level of injury as well as inability to replace lost neural cells and to stimulate neurogenesis. In contrast, fish and amphibians are capable of regenerating a variety of their organs like limb/fin, jaw, heart and ...
More >>
MPM-2 Mediated Immunoprecipitation of Proteins Undergoing Proline-directed Phosphorylation
Authors:
Roberta Antonelli
and
Paola Zacchi
,
date:
12/05/2016,
view:
3589,
Q&A:
0
Immunoprecipitation (IP) represents a widely utilized biochemical method to isolate a specific protein from a complex mixture taking advantage of an antibody that specifically recognizes that particular target molecule. This procedure is extremely versatile and can be applied to concentrate a specific protein, to identify interacting partners in ...
More >>
Delayed Spatial Win-shift Test on Radial Arm Maze
Authors:
Simone N. De Luca
,
Luba Sominsky
and
Sarah J. Spencer
,
date:
12/05/2016,
view:
4672,
Q&A:
0
The radial arm maze (RAM) is used to assess reference and working memory in rodents. This task relies on the rodent’s ability to orientate itself in the maze using extra-maze visual cues. This test can be used to investigate whether a rodent’s cognition is improved or impaired under a variety of experimental conditions. Here, we describe one way ...
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Plant Science
Isolating and Measuring the Growth and Morphology of Pro-embryogenic Masses in
Araucaria angustifolia
(Bertol.) Kuntze (Araucariaceae)
Authors:
Jackellinne Caetano Douétts-Peres
,
Vanildo Silveira
,
Marco Antonio Lopes Cruz
and
Claudete Santa-Catarina
,
date:
12/05/2016,
view:
3362,
Q&A:
0
Embryogenic suspension cultures of
Araucaria angustifolia
(Bertol.) Kuntze (Araucariaceae) can be used as a model to test the effects of compounds added to the culture medium on the cellular growth and morphology of Pro-Embryogenic Masses (PEMs). PEMs are formed by embryogenic and suspensor-type cells. To measure changes in the cellular ...
More >>
Microplate Assay to Study Carboxypeptidase A Inhibition in Andean Potatoes
Authors:
Mariana Edith Tellechea
,
Javier Garcia-Pardo
,
Juliana Cotabarren
,
Daniela Lufrano
,
Laura Bakas
,
Francesc Xavier Avilés
,
Walter David Obregon
,
Julia Lorenzo
and
Sebastián Tanco
,
date:
12/05/2016,
view:
3896,
Q&A:
0
Metallocarboxypeptidases (MCP) are zinc-dependent exopeptidases that catalyze the hydrolysis of C-terminal amide bonds in proteins and peptides. They are involved in a wide range of physiological processes and have recently emerged as relevant drug targets in biomedicine (Arolas
et al.
, 2007). In this context, the study and discovery of ...
More >>
Extraction and Measurement of Abscisic Acid in a Unicellular Red Alga
Cyanidioschyzon merolae
Authors:
Yuki Kobayashi
and
Kan Tanaka
,
date:
12/05/2016,
view:
3814,
Q&A:
0
Abscisic acid (ABA) has been known as a phytohormone of land plants, which is synthesized in response to abiotic stresses and induces various physiological responses, but is also found from eukaryotic algae. Recently, we reported that a unicellular red alga
Cyanidioschyzon merolae
produced ABA, which prevented cell growth and enhanced ...
More >>
Fusarium graminearum
Maize Stalk Infection Assay and Associated Microscopic Observation Protocol
Authors:
Juan He
,
Tinglu Yuan
and
Wei-Hua Tang
,
date:
12/05/2016,
view:
4310,
Q&A:
0
The ascomycete fungus
Fusarium graminearum
(previously also called
Gibberella zeae
) causes
Gibberella
stalk rot in maize (
Zea mays
) and results in lodging and serious yield reduction. To develop methods to assess the fungal growth and symptom development in maize stalks, we present here a protocol of maize stalk ...
More >>
Measurement of ATP Hydrolytic Activity of Plasma Membrane H
+
-ATPase from
Arabidopsis thaliana
Leaves
Authors:
Masaki Okumura
and
Toshinori Kinoshita
,
date:
12/05/2016,
view:
3513,
Q&A:
0
Plant plasma membrane H
+
-ATPase, which is a P-type ATPase, couples ATP hydrolysis to H
+
extrusion and thereby generates an electrochemical gradient across the plasma membrane. The proton gradient is necessary for secondary transport, cell elongation, and membrane potential maintenance. Here we describe a protocol for ...
More >>
Shoot Apical Meristem Size Measurement
Authors:
Hsuan Chou
,
Huanzhong Wang
and
Gerald A. Berkowitz
,
date:
12/05/2016,
view:
3484,
Q&A:
0
The shoot apical meristem (SAM) is a collection of cells that continuously renew themselves by cell division and also provide cells to newly developing organs. It has been known that CLAVATA (CLV) 3 peptide regulates a transcription factor WUSCHEL (WUS) to keep numbers of undifferentiated cells constant and maintain the size of the SAM. The ...
More >>
A Golden Gate-based Protocol for Assembly of Multiplexed gRNA Expression Arrays for CRISPR/Cas9
Authors:
Johan Vad-Nielsen
,
Lin Lin
,
Kristopher Torp Jensen
,
Anders Lade Nielsen
and
Yonglun Luo
,
date:
12/05/2016,
view:
7123,
Q&A:
1
The CRISPR (clustered regularly interspaced short palindromic repeats)-associated protein 9 (Cas9) has become the most broadly used and powerful tool for genome editing. Many applications of CRISPR-Cas9 require the delivery of multiple small guide RNAs (gRNAs) into the same cell in order to achieve multiplexed gene editing or regulation. Using ...
More >>
Tandem Purification of His
6
-3x FLAG Tagged Proteins for Mass Spectrometry from
Arabidopsis
Authors:
He Huang
and
Dmitri Anton Nusinow
,
date:
12/05/2016,
view:
4980,
Q&A:
0
Tandem affinity purification is a powerful method to identify protein complexes that function in association with a known gene of interest. This protocol describes a methodology to capture proteins tagged with His
6
-3x FLAG explicitly for the purpose of on-bead digestion and identification by mass spectrometry. The high sensitivity and ...
More >>
Stem Cell
In vitro
Chondrogenic Hypertrophy Induction of Mesenchymal Stem Cells
Authors:
Sang Young Jeong
,
Miyoung Lee
,
Soo Jin Choi
,
Wonil Oh
and
Hong Bae Jeon
,
date:
12/05/2016,
view:
4403,
Q&A:
0
To investigate underlying mechanism of chondrogenic hypertrophy, we need proper
in vitro
hypertrophic model of mesenchymal stem cells (MSCs). This protocol describes our defined method for induction of
in vitro
chondrogenic hypertrophy of human umbilical cord blood-derived MSCs (hUCB-MSCs). By adding thyroid hormone (T3; ...
More >>
Systems Biology
Protocol for Molecular Dynamics Simulations of Proteins
Authors:
MNV Prasad Gajula
,
Anuj Kumar
and
Johny Ijaq
,
date:
12/05/2016,
view:
8243,
Q&A:
0
Molecular dynamics (MD) simulations have become one of the most important tools in understanding the behavior of bio-molecules on nanosecond to microsecond time scales. In this protocol, we provide a general approach and standard setup protocol for MD simulations by using the Gromacs MD suite.
More >>