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Latest Articles

Using 3dRNA for RNA 3‐D Structure Prediction and Evaluation
Jian Wang, Yi Xiao
This unit describes how to use 3dRNA to predict RNA 3‐D structures from their sequences and secondary (2‐D) structures, and how to use 3dRNAscore to evaluate the predicted structures. The predicted RNA 3‐D structures can be used to predict or understand their functions and can also be used to find
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Phylogenetic Inference Using RevBayes
Sebastian Höhna, Michael J. Landis, Tracy A. Heath
Bayesian phylogenetic inference aims to estimate the evolutionary relationships among different lineages (species, populations, gene families, viral strains, etc.) in a model‐based statistical framework that uses the likelihood function for parameter estimates. In recent years, evolutionary models
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Using FunSeq2 for Coding and Non‐Coding Variant Annotation and Prioritization
Priyanka Dhingra, Yao Fu, Mark Gerstein, Ekta Khurana
The identification of non‐coding drivers remains a challenge and bottleneck for the use of whole‐genome sequencing in the clinic. FunSeq2 is a computational tool for annotation and prioritization of somatic mutations in coding and non‐coding regions. It integrates a data context made from
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Using NCBI BLAST
Naomi A. Stover, Andre R.O. Cavalcanti
BLAST is the most widely used software in bioinformatics research. Its main function is to compare a sequence of interest, the query sequence, to sequences in a large database. BLAST then reports the best matches, or “hits,” found in the database. This simple program has two primary applications.
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Beyond Simple Homology Searches: Multiple Sequence Alignments and Phylogenetic Trees
Rebecca A. Zufall
Phylogenetic trees represent hypotheses about evolutionary relationships between organisms or nucleotide or amino acid sequences. Because the best BLAST hit often does not represent the most closely related sequence, phylogenetic analyses are an essential extension of inquiry into any new protein or
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Practical Considerations When Altering Digital Images
Jerry Sedgewick
Worldwide, Adobe Photoshop is the primary means in research for altering images. Training on the use of this powerful tool most often is peer to peer within a laboratory, or it is self‐taught through trial and error. As a result, varying amounts of visual data can be removed unwittingly; contrast‐
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Statistical Analyses
Johanna Hardin, John Kloke
In this appendix, we provide an outline of methods used in analyzing biological data. We give a summary of types of data encountered and the appropriate methods to apply for the questions of interest. Statistical techniques described include the t test, the Wilcoxon rank sum test, the
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Burkholderia thailandensis: Genetic Manipulation
Erin C. Garcia
Burkholderia thailandensis is a Gram‐negative bacterium endemic to Southeast Asian and northern Australian soils. It is non‐pathogenic; therefore, it is commonly used as a model organism for the related human pathogens Burkholderia mallei and Burkholderia pseudomallei . B. thailandensis is
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Lyophilization of Bdellovibrio bacteriovorus 109J for Long‐Term Storage
Mélanie J. Boileau, Rinosh Mani, Kenneth D. Clinkenbeard
Bdellovibrio bacteriovorus 109J is a Gram‐negative predatory bacterium with obligate host dependency on other Gram‐negative bacteria. This bacteriolytic predator collides with, enters, and establishes growth within the prey (host) periplasm, eventually lysing the prey cell wall to release fresh,
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Chlamydia trachomatis Transformation and Allelic Exchange Mutagenesis
Konrad E. Mueller, Katerina Wolf, Kenneth A. Fields
Gene inactivation is essential for forward and reverse genetic approaches to establish protein function. Techniques such as insertion or chemical mutagenesis have been developed to mutagenize chlamydiae via targeted or random mutagenesis, respectively. Both of these approaches require transformation
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Analysis of Human Papillomavirus Genome Replication Using Two‐ and Three‐Dimensional Agarose Gel Electrophoresis
Liisi Henno, Eva‐Maria Tombak, Jelizaveta Geimanen, Marit Orav, Ene Ustav, Mart Ustav
This unit includes the necessary information to conduct neutral/neutral and neutral/alkaline two‐dimensional and neutral/neutral/alkaline three‐dimensional agarose gel electrophoresis. The methodology has been optimized over the years to gain a better outcome from the hard‐to‐interpret signals of
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Purification Toxoplasma gondii Tissue Cysts Using Percoll Gradients
Elizabeth A. Watts, Animesh Dhara, Anthony P. Sinai
The protozoan parasite Toxoplasma gondii is capable of infecting all warm‐blooded animals and humans. Infectious, transmissible forms of the parasite include oocysts produced by the sexual cycle within the definitive feline host and tissue cysts that form Toxoplasma in the central nervous system and
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Isolation, Culture and Cryopreservation of Sarcocystis species
S. K. Verma, D. S. Lindsay, M. E. Grigg, J. P. Dubey
More than 200 valid Sarcocystis species have been described in the parasitological literature. The developmental life cycle in the intermediate host and definitive host has only been described for a few species. Sarcocystis parasites are common pathogens infecting a wide range of animals, including
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Isolation of Stem Cells and Progenitors from Mouse Epidermis
Lana Kostic, Egor Sedov, Despina Soteriou, Yahav Yosefzon, Yaron Fuchs
The epidermis consists of several distinct compartments including the interfollicular epidermis (IFE), sweat glands, sebaceous glands (SGs), and the hair follicle (HF). While the IFE and SGs are in a constant state of self‐renewal, the HF cycles between phases of growth, destruction, and rest. The
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Isolation of Ready‐to‐Use Adipose‐Derived Stem Cell (ASC) Pellet for Clinical Applications and a Comparative Overview of Alternate Methods for ASC Isolation
Edoardo Raposio, Nicolò Bertozzi
Current literature does not offer a standardized method to isolate adipose‐derived stem cells (ASCs) for clinical applications and hence clinical studies using ASCs often show inconsistent results. Most of these studies borrow laboratory or benchside‐derived protocols, which are complex, time
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Human Umbilical Cord Mesenchymal Stromal Cell Isolation, Expansion, Cryopreservation, and Characterization
J. Robert Smith, Adrienne Cromer, Mark L. Weiss
Revised methods to derive, expand, and characterize mesenchymal stromal cells (MSCs) from the umbilical cord are provided. Several considerations are taken for GMP compliance including using a closed system isolation method and eliminating several xenogenic components. With this method cells are
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Angiogenesis within Stem Cell–Seeded Silk Scaffolds Cultured on the Chorioallantoic Membrane and Visualized by 3D Imaging
Anna Woloszyk, Thimios A. Mitsiadis
The long‐term survival and successful integration of implants for tissue replacement and regeneration highly depends upon the fast ingrowth of blood vessels from the surrounding tissues. Before selecting potential biomaterials for clinical applications, they must be thoroughly tested with proper
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Rhesus Macaque iPSC Generation and Maintenance
Ravi Chandra Yada, So Gun Hong, Yongshun Lin, Thomas Winkler, Cynthia E. Dunbar
The rhesus macaque ( Macaca mulatta ) is physiologically and phylogenetically similar to humans, and therefore represents an invaluable model for the pre‐clinical assessment of the safety and feasibility of iPSC‐derived cell therapies. The use of an excisable polycistronic lentiviral STEMCCA vector
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Tetracycline‐Inducible and Reversible Stable Gene Expression in Human iPSC‐Derived Neural Progenitors and in the Postnatal Mouse Brain
Aslam Abbasi Akhtar, Joshua J. Breunig
The pB‐tet‐GOI plasmid system allows for stable piggyBac transposition‐mediated integration into cells, a fluorescent nuclear reporter to identify cells that have been transfected, and robust transgene activation or suppression upon the addition of dox to the cell culture or diet of the animal.
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Quantification of Lactate Dehydrogenase for Cell Viability Testing Using Cell Lines and Primary Cultured Astrocytes
Simon Kaja, Andrew J. Payne, Yuliya Naumchuk, Peter Koulen
Drug discovery heavily relies on cell viability studies to assess the potential toxicity of drug candidates. L ‐Lactate dehydrogenase (LDH) is a cytoplasmic enzyme that catalyzes the concomitant interconversions of pyruvate to L ‐lactate and NADH to NAD + during glycolysis, and the reverse reactions
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Using Chromatin Immunoprecipitation in Toxicology: A Step‐by‐Step Guide to Increasing Efficiency, Reducing Variability, and Expanding Applications
Shaun D. McCullough, Doan M. On, Emma C. Bowers
Histone modifications work in concert with DNA methylation to regulate cellular structure, function, and response to environmental stimuli. More than 130 unique histone modifications have been described to date, and chromatin immunoprecipitation (ChIP) allows for the exploration of their
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Behavioral Phenotyping for Autism Spectrum Disorders in Mice
Yu‐Chi Chang, Toby B. Cole, Lucio G. Costa
Autism spectrum disorder (ASD) represents a heterogeneous group of disorders characterized by alterations in three behavioral symptom domains: Social interactions, verbal and nonverbal communication, and repetitive behaviors. Increasing prevalence of ASD in recent years suggests that exposure to
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A Multi‐Parametric Fluorescent Assay for the Screening and Mechanistic Study of Drug‐Induced Steatosis in Liver Cells in Culture
Laia Tolosa, M. José Gómez‐Lechón, M. Teresa Donato
Human hepatic cells have been used for drug safety risk evaluations throughout early development phases. They provide rapid, cost‐effective early feedback to identify drug candidates with potential hepatotoxicity. This unit presents a cell‐based assay to evaluate the risk of liver damage associated
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Use of Primary Rat Hepatocytes for Prediction of Drug‐Induced Mitochondrial Dysfunction
Cong Liu, Shuichi Sekine, Binbin Song, Kousei Ito
Mitochondrial dysfunction plays a central role in drug‐induced liver injury. To evaluate drug‐induced mitochondrial impairment, several isolated mitochondria‐ or cell line‐based assays have been reported. Among them, culturing HepG2 cells in galactose provides a remarkable method to assess
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Micropatterned Co‐Cultures of Human Hepatocytes and Stromal Cells for the Assessment of Drug Clearance and Drug‐Drug Interactions
Christine Lin, Salman R Khetani
Drug clearance rates from the body can determine drug exposure that can affect efficacy or toxicity. Thus, accurate prediction of drug clearance during preclinical development can help guide dose selection in humans, but animal testing is not always predictive of human outcomes. Because hepatic drug
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