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Volume Measurement
Thomas Davis, Andrew Zanella
This unit describes the common types of volumetric apparatus used in the life science laboratory, their use, and care. When an experimenter needs to prepare solutions at accurate concentrations and quantitatively transfer samples of liquid from one container to another, an array of glassware and
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Fluorescence Spectroscopy
Claudia Y. Lee
Fluorescence is an extremely powerful tool in modern biology, physics, and chemistry laboratories. This unit begins with the physics of fluorescence, the biological applications of fluorescence, and the mechanisms behind spectrometers and fluorometers, followed by strategies to choose an appropriate
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Mammalian Cell Culture
Dennis R. Warner, Daisuke Sakai, Lisa L. Sandell
Mammalian cell culture is the process of growing animal cells in vitro in a flask or dish. This unit describes the methods, equipment, supplies, and reagents used in a cell culture laboratory. Because preventing contamination and maintaining purity of cell cultures is arguably the greatest challenge
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Agarose Gel Electrophoresis
Jennifer A. Armstrong, Joseph R. Schulz
Agarose gel electrophoresis, which separates and sizes linear DNA and RNA fragments, is arguably the most basic and essential technique in molecular biology. It is commonly employed for analysis of PCR products, plasmid DNA, and products of restriction enzyme digestion. It is the first step for
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Immunofluorescence Microscopy
David J. Asai
The visualization of fluorescently tagged molecules is a powerful strategy that can contribute to the understanding of the complex dynamics of the cell. A particularly robust and broadly applicable method is immunofluorescence microscopy, in which a specific fluorescently labeled antibody binds the
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Isolating Viral and Host RNA Sequences from Archival Material and Production of cDNA Libraries for High‐Throughput DNA Sequencing
Yongli Xiao, Zong‐Mei Sheng, Jeffery K. Taubenberger
The vast majority of surgical biopsy and post‐mortem tissue samples are formalin‐fixed and paraffin‐embedded (FFPE), but this process leads to RNA degradation that limits gene expression analysis. As an example, the viral RNA genome of the 1918 pandemic influenza A virus was previously determined in
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Genetic Manipulation of Stenotrophomonas maltophilia
Elliott Welker, Yayra Domfeh, Deepti Tyagi, Sanjivni Sinha, Nathan Fisher
Stenotrophomonas maltophilia is a Gram‐negative, aerobic, motile, environmental bacterium that is emerging as an important nosocomial pathogen with high rates of attributable mortality in severely ill patients. S. maltophilia is of particular concern to patients suffering from cystic fibrosis (CF)
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Fluorescently Labeled Human Papillomavirus Pseudovirions for Use in Virus Entry Experiments
Pilar Samperio Ventayol, Mario Schelhaas
Human papillomaviruses (HPV) infect skin or mucosal epidermis. The simplistic capsid consists of a major capsid protein L1, a minor capsid protein L2, and a double‐stranded circular DNA of about 8kB in size. The development of HPV‐based vectors [i.e., pseudovirions (PsV)] as tools to study the
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Rotaviruses: Extraction and Isolation of RNA, Reassortant Strains, and NSP4 Protein
Krystle A. Yakshe, Zachary D. Franklin, Judith M. Ball
Rotavirus (RV) contains 11 double‐stranded RNA segments that encode for twelve structural and nonstructural proteins. The separation and isolation of viral RNA is a necessary precursor for many experimental techniques and can be useful for rapid RV RNA typing and sequencing of different rotavirus
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Growth and Quantification of MERS‐CoV Infection
Christopher M. Coleman, Matthew B. Frieman
Middle East respiratory syndrome coronavirus (MERS‐CoV) is an emerging highly pathogenic respiratory virus. Although MERS‐CoV only emerged in 2012, we and others have developed assays to grow and quantify infectious MERS‐CoV and RNA products of replication in vitro. MERS‐CoV is able to infect a
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Isolation and Expansion of Mesenchymal Stem Cells from Human Conjunctival Tissue
Samad Nadri, Shahin Yazdani
Here we describe a simple protocol for the isolation and culture of mesenchymal stem cells (MSCs) from conjunctiva stromal tissue, with applications to stem cell biology and regenerative medicine. This protocol is based on an explant culture protocol for the adhesion and migration of MSCs from
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Neural Crest Cells from Dual SMAD Inhibition
Faranak Fattahi, Lorenz Studer, Mark J. Tomishima
Neural crest (NC) cells are migratory multipotent progenitors that delaminate from the neural tube during embryonic development and give rise to various cell types in different organs. These cells are a transient embryonic cell population and therefore difficult to obtain from primary sources.
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Quantification of Glutathione in Caenorhabditis elegans
Samuel W. Caito, Michael Aschner
Glutathione (GSH) is the most abundant intracellular thiol with diverse functions from redox signaling, xenobiotic detoxification, and apoptosis. The quantification of GSH is an important measure for redox capacity and oxidative stress. This protocol quantifies total GSH from Caenorhabditis elegans
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High‐Resolution Multi‐Photon Imaging of Morphological Structures of Caenorhabditis elegans
Gabriele M. Bixel, Stephanie J.B. Fretham, Michael Aschner
In this protocol, we combine two‐photon excitation fluorescence with nonlinear optical measurements to reconstruct the three‐dimensional architecture of the pharyngeal region and the muscular system of the anterior and mid‐body region of Caenorhabditis elegans ( C. elegans ). Femto‐second laser
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Detection of Bulky Endogenous Oxidative DNA Lesions Derived from 8,5′‐Cyclo‐2′‐deoxyadenosine by 32P‐Postlabeling Assay
Guo‐Dong Zhou, Bhagavatula Moorthy
8,5′‐Cyclopurine‐2′‐deoxynucleotides represent a class of oxidative DNA lesions that are specifically repaired by nucleotide excision repair but not by base excision repair or direct enzymatic reversion. The 32 P‐postlabeling assay is an ultrasensitive method that has been extensively used for the
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microRNA Profiling as Tool for Developmental Neurotoxicity Testing (DNT)
Lena Smirnova, Andrea E.M. Seiler, Andreas Luch
microRNAs (miRNAs) are small non‐coding RNA molecules functioning as post‐transcriptional regulators of gene expression. miRNAs play a significant role in organism development, regulating developmental timing, cell differentiation, and specification. In the developing brain, miRNAs regulate neural
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