18,000 peer-reviewed, regularly
updated laboratory procedures
Cutting-edge protocols developed
by leading research scientists
Indexed in
PubMed and Scopus

Latest Articles

Method for DNA Ploidy Analysis Along with Immunophenotyping for Rare Populations in a Sample using FxCycle Violet
Prashant Tembhare, Yajamanam Badrinath, Sitaram Ghogale, Papagudi Ganesan Subramanian
The clinical use of flow cytometric DNA ploidy assay has been extended towards stratifying the risk of diseases, such as monoclonal gammopathies or B cell acute lymphoblastic leukemia, and to detect circulating tumor cells, both of which require detection of minute cell populations. This unit
Abstract | Full Text: HTML PDF
Fluorescent Proteins for Flow Cytometry
Teresa S. Hawley, Robert G. Hawley, William G. Telford
Fluorescent proteins have become standard tools for cell and molecular biologists. The color palette of fluorescent proteins spans the ultraviolet, visible, and near‐infrared spectrum. Utility of fluorescent proteins has been greatly facilitated by the availability of compact and affordable solid
Abstract | Full Text: HTML PDF
Method to Detect the Cellular Source of Over‐Activated NADPH Oxidases Using NAD(P)H Fluorescence Lifetime Imaging
Daniel Bremer, Ruth Leben, Ronja Mothes, Helena Radbruch, Raluca Niesner
Fluorescence‐lifetime imaging microscopy (FLIM) is a technique to generate images, in which the contrast is obtained by the excited‐state lifetime of fluorescent molecules instead of their intensity and emission spectrum. The ubiquitous coenzymes NADH and NADPH, hereafter NAD(P)H, in cells show a
Abstract | Full Text: HTML PDF
High‐Throughput Particle Uptake Analysis by Imaging Flow Cytometry
Asya Smirnov, Michael D. Solga, Joanne Lannigan, Alison K. Criss
Quantifying the efficiency of particle uptake by host cells is important in the fields of infectious diseases, autoimmunity, cancer, developmental biology, and drug delivery. Here we present a protocol for high‐throughput analysis of particle uptake by imaging flow cytometry, using the bacterium
Abstract | Full Text: HTML PDF
Correlative Fluorescence and Electron Microscopy in 3D—Scanning Electron Microscope Perspective
Jonathan Franks, Callen T. Wallace, Masateru Shibata, Mitsuo Suga, Natasha Erdman, Donna B. Stolz, Simon C. Watkins
The ability to correlate fluorescence microscopy (FM) and electron microscopy (EM) data obtained on biological (cell and tissue) specimens is essential to bridge the resolution gap between the data obtained by these different imaging techniques. In the past such correlations were limited to either
Abstract | Full Text: HTML PDF
Overview of Genetic Diagnosis in Cancer
Bruce R. Korf, Fady M. Mikhail
Both cytogenetic and molecular genetic studies can contribute to the management of patients with cancer. In some cases, genetic markers are specific to particular tumor types and are useful in diagnosis. This can be helpful in distinguishing histologically similar tumors that may respond differently
Abstract | Full Text: HTML PDF
Molecular Analysis of Genetic Markers for Non‐Hodgkin Lymphomas
Lynette M. Sholl, Janina Longtine, Frank C. Kuo
Molecular analysis complements the clinical and histopathologic tools used to diagnose and subclassify hematologic malignancies. The presence of clonal antigen‐receptor gene rearrangements can help to confirm the diagnosis of a B or T cell lymphoma and can serve as a fingerprint of that neoplasm to
Abstract | Full Text: HTML PDF
Computational Approach to Measuring Myocyte Disarray in Animal Models of Heart Disease
William Wan, Leslie Leinwand
In cardiovascular disease research, studies often include measuring cardiac function and performing histological examination of heart tissue. After measuring contractility, hearts from animals such as mice and rats are often frozen or fixed, sliced, and stained to quantify the morphology of various
Abstract | Full Text: HTML PDF
High‐Risk Screening for Fabry Disease: Analysis by Tandem Mass Spectrometry of Globotriaosylceramide (Gb3) in Urine Collected on Filter Paper
Christiane Auray‐Blais, Pamela Lavoie, Michel Boutin, Mona Abaoui
Fabry disease is a complex, panethnic lysosomal storage disorder. It is characterized by the accumulation of glycosphingolipids in tissues, organs, the vascular endothelium, and biological fluids. The reported incidence in different populations is quite variable, ranging from 1:1400 to 1:117,000.
Abstract | Full Text: HTML PDF
Methods for Quantitative Creatinine Determination
John F. Moore, J. Daniel Sharer
Reliable measurement of creatinine is necessary to assess kidney function, and also to quantitate drug levels and diagnostic compounds in urine samples. The most commonly used methods are based on the Jaffe principal of alkaline creatinine‐picric acid complex color formation. However, other
Abstract | Full Text: HTML PDF
Basic Multicolor Flow Cytometry
Zofia Maciorowski, Pratip K. Chattopadhyay, Paresh Jain
Multicolor flow cytometry is a rapidly evolving technology that uses multiple fluorescent markers to identify and characterize cellular subpopulations of interest, allowing rapid analysis on tens of thousands of cells per second, with the possibility of isolating pure, viable populations by cell
Abstract | Full Text: HTML PDF
Measurement of Tumor Necrosis Factor and Lymphotoxins
M. Michele Hogan, Stefanie N. Vogel
The tumor necrosis factor (TNF) superfamily of cytokines plays critical roles in all aspects of the immune response. TNF and the lymphotoxins (LT), LTα and LTβ, are particularly important as major effector cytokines and mediators or lymphoid organ development. One of the classical methods for
Abstract | Full Text: HTML PDF
Transfection by Electroporation
Huntington Potter, Richard Heller
Electroporation—the use of high‐voltage electric shocks to introduce DNA into cells—can be used with most cell types, yields a high frequency of both stable transformation and transient gene expression, and, because it requires fewer steps, can be easier than alternative techniques. This unit
Abstract | Full Text: HTML PDF
Characterization and Functional Analysis of Mouse Semi‐invariant Natural T Cells
Amrendra Kumar, Jelena S. Bezbradica, Aleksandar K. Stanic, Sebastian Joyce
Semi‐invariant natural killer T (iNKT) cells are CD1d‐restricted innate‐like lymphocytes that recognize lipid agonists. Activated iNKT cells have immunoregulatory properties. Human and mouse iNKT cell functions elicited by different glycolipid agonists are highly conserved, making the mouse an
Abstract | Full Text: HTML PDF
Gut Microbiome Standardization in Control and Experimental Mice
Kathy D. McCoy, Markus B. Geuking, Francesca Ronchi
Mouse models are used extensively to study human health and to investigate the mechanisms underlying human disease. In the past, most animal studies were performed without taking into consideration the impact of the microbiota. However, the microbiota that colonizes all body surfaces, including the
Abstract | Full Text: HTML PDF
Direct Isolation of Seamless Mutant Bacterial Artificial Chromosomes
George T. Lyozin, Yasuhiro Kosaka, Gourab Bhattacharje, H. Joseph Yost, Luca Brunelli
Seamless (i.e., without unwanted DNA sequences) mutant bacterial artificial chromosomes (BACs) generated via recombination‐mediated genetic engineering (recombineering) are better suited to study gene function compared to complementary DNA (cDNA) because they contain only the specific mutation and
Abstract | Full Text: HTML PDF
Mapping Transposon Insertions in Bacterial Genomes by Arbitrarily Primed PCR
José T. Saavedra, Julia A. Schwartzman, Michael S. Gilmore
Transposons can be used to easily generate and label the location of mutations throughout bacterial and other genomes. Transposon insertion mutants may be screened for a phenotype as individual isolates, or by selection applied to a pool of thousands of mutants. Identifying the location of a
Abstract | Full Text: HTML PDF
Analysis of Membrane Protein Interactions with a Bacterial Adenylate Cyclase–Based Two‐Hybrid (BACTH) Technique
Scot P. Ouellette, Gouzel Karimova, Marilyne Davi, Daniel Ladant
The bacterial two‐hybrid (BACTH, for “Bacterial Adenylate Cyclase‐based Two‐Hybrid”) technique is a simple and fast genetic approach to analyze protein‐protein interactions in vivo. In this system, the proteins of interest are genetically fused to two complementary fragments from the catalytic
Abstract | Full Text: HTML PDF
Preparing Viable Single Cells from Human Tissue and Tumors for Cytomic Analysis
Nalin Leelatian, Deon B. Doxie, Allison R. Greenplate, Justine Sinnaeve, Rebecca A. Ihrie, Jonathan M. Irish
Mass cytometry is a single‐cell biology technique that samples >500 cells per second, measures >35 features per cell, and is sensitive across a dynamic range of >10 4 relative intensity units per feature. This combination of technical assets has powered a series of recent cytomic studies
Abstract | Full Text: HTML PDF
Transcriptome‐wide Identification of RNA‐binding Protein Binding Sites Using Photoactivatable‐Ribonucleoside‐Enhanced Crosslinking Immunoprecipitation (PAR‐CLIP)
Henrike Maatz, Marcin Kolinski, Norbert Hubner, Markus Landthaler
RNA‐binding proteins (RBPs) mediate important co‐ and post‐transcriptional gene regulation by binding pre‐mRNA in a sequence‐ and/or structure‐specific manner. For a comprehensive understanding of RBP function, transcriptome‐wide mapping of the RNA‐binding sites is essential, and CLIP‐seq methods
Abstract | Full Text: HTML PDF
WGA‐Alexa Conjugates for Axonal Tracing
Sabrina L. Levy, Joshua J. White, Elizabeth P. Lackey, Lindsey Schwartz, Roy V. Sillitoe
Anatomical labeling approaches are essential for understanding brain organization. Among these approaches are various methods of performing tract tracing. However, a major hurdle to overcome when marking neurons in vivo is visibility. Poor visibility makes it challenging to image a desired neuronal
Abstract | Full Text: HTML PDF
Fluorescence Microscopy: A Concise Guide to Current Imaging Methods
Christian A. Combs, Hari Shroff
The field of fluorescence microscopy is rapidly growing and offers ever more imaging capabilities for biologists. Over the past decade, many new technologies and techniques have been developed that allow for combinations of deeper, faster, and higher resolution imaging. These have included the
Abstract | Full Text: HTML PDF
Time‐Lapse Imaging and Cell Tracking of Migrating Cells in Slices and Flattened Telencephalic Vesicles
Verónica Murcia‐Belmonte, Giovanna Expósito, Eloísa Herrera
Neuronal migration is a vital process needed for subsequent assembly and function of neural circuitry during embryonic development. The vast majority of neural progenitors are generated far from their final destination and need to migrate considerable distances to reach their specific cortical
Abstract | Full Text: HTML PDF
CRISPR/Cas9‐Mediated Gene Knockout in the Mouse Brain Using In Utero Electroporation
Yohei Shinmyo, Hiroshi Kawasaki
This unit describes a highly efficient and rapid procedure for brain‐specific disruption of genes in the developing mouse brain using pX330 plasmids expressing humanized Cas9 and single‐guide RNAs (sgRNAs) against target genes. The pX330 plasmids are delivered into the rodent brain using in utero
Abstract | Full Text: HTML PDF
Quantitative High‐Throughput Screening Using a Coincidence Reporter Biocircuit
Brittany W. Schuck, Ryan MacArthur, James Inglese
Reporter‐biased artifacts—i.e., compounds that interact directly with the reporter enzyme used in a high‐throughput screening (HTS) assay and not the biological process or pharmacology being interrogated—are now widely recognized to reduce the efficiency and quality of HTS used for chemical probe
Abstract | Full Text: HTML PDF
Systems Genetic Analysis in GeneNetwork.org
Clarissa C. Parker, Price E. Dickson, Vivek M. Philip, Mary Thomas, Elissa J. Chesler
Genome‐wide association studies (GWAS) have emerged as a powerful tool to identify alleles and molecular pathways that influence susceptibility to psychiatric disorders and other diseases. Forward genetics using mouse mapping populations allows for a complementary approach that provides rigorous
Abstract | Full Text: HTML PDF
Fluorescein Isothiocyanate (FITC)‐Dextran Extravasation as a Measure of Blood‐Brain Barrier Permeability
Reka Natarajan, Nicole Northrop, Bryan Yamamoto
The blood‐brain barrier (BBB) is formed in part by vascular endothelial cells that constitute the capillaries and microvessels of the brain. The function of this barrier is to maintain homeostasis within the brain microenvironment and buffer the brain from changes in the periphery. A dysfunction of
Abstract | Full Text: HTML PDF
Computational Prediction of Intrinsic Disorder in Proteins
Fanchi Meng, Vladimir Uversky, Lukasz Kurgan
Computational prediction of intrinsically disordered proteins (IDPs) is a mature research field. These methods predict disordered residues and regions in an input protein chain. More than 60 predictors of IDPs have been developed. This unit defines computational prediction of intrinsic disorder,
Abstract | Full Text: HTML PDF
N‐Terminal Methionine Processing
Paul T. Wingfield
Protein synthesis is initiated by methionine in eukaryotes and by formylmethionine in prokaryotes. N‐terminal methionine can be co‐translationally cleaved by the enzyme methionine aminopeptidase (MAP). When recombinant proteins are expressed in bacterial and mammalian expression systems, there is a
Abstract | Full Text: HTML PDF
Immunoblotting and Immunodetection
Duojiao Ni, Peng Xu, Sean Gallagher
Immunoblotting (western blotting) is used to identify specific antigens recognized by polyclonal or monoclonal antibodies. This unit provides protocols for all steps, starting with solubilization of the protein samples, usually by means of SDS and reducing agents. Following solubilization, the
Abstract | Full Text: HTML PDF
Selective Proteomic Proximity Labeling Assay Using Tyramide (SPPLAT): A Quantitative Method for the Proteomic Analysis of Localized Membrane‐Bound Protein Clusters
Johanna Susan Rees, Xue‐Wen Li, Sarah Perrett, Kathryn Susan Lilley, Antony Philip Jackson
This manuscript describes a new and general method to identify proteins localized into spatially restricted membrane microenvironments. Horseradish peroxidase (HRP) is brought into contact with a target protein by being covalently linked to a primary or secondary antibody, an antigen or substrate, a
Abstract | Full Text: HTML PDF
Bioluminescence Resonance Energy Transfer (BRET)‐Based Synthetic Sensor Platform for Drug Discovery
Jongchan Woo, Jason Hong, Savithramma P. Dinesh‐Kumar
Bioluminescence resonance energy transfer (BRET) is a technique that analyzes protein‐protein interactions (PPIs). The unique feature of BRET delineates that the resonance energy is generated by the resonance energy donor, Renilla luciferase by the oxidative decarboxylation of coelenterazine
Abstract | Full Text: HTML PDF
Measuring Protein Interactions by Optical Biosensors
Huaying Zhao, Lisa F. Boyd, Peter Schuck
This unit gives an introduction to the basic techniques of optical biosensing for measuring equilibrium and kinetics of reversible protein interactions. Emphasis is placed on description of robust approaches that will provide reliable results with few assumptions. How to avoid the most commonly
Abstract | Full Text: HTML PDF