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

Latest Articles

Weight Measurement
Andrew Hurdle, Michael Guzy
This unit provides information to aid in the selection and proper use of a laboratory balance. Laboratory balances are used for measuring the mass of an object and come in two main types: mechanical and electronic. Electronic balances generally come with a computer interface to facilitate the
Abstract | Full Text: HTML PDF
Rob Morris
Spectroscopy is the study of the interaction of light with matter. By observing how light interacts with matter—through reflection, refraction, elastic scattering, absorbance, inelastic scattering, and emission—it is possible to (1) identify the wavelengths of light that interact with atoms and
Abstract | Full Text: HTML PDF
Overview of qPCR Molecular Probes
Andrei Laikhter
In recent times, varieties of modified synthetic oligonucleotides conjugated with reporter molecules have been employed in hybridization‐based assays. These methods have broad applications in both basic molecular biology research and in clinical diagnostics and screening because they have high
Abstract | Full Text: HTML PDF
Ethical Considerations When Altering Digital Images
Sean R. Gallagher, Joseph Neuman
The ubiquitous use of digital imaging has greatly simplified and accelerated life science research. However, the ease of manipulating digital images also presents serious ethical issues. This unit presents guidelines and cautions describing best practices for use, analysis, and presentation of
Abstract | Full Text: HTML PDF
Isolation and Functional Analysis of Human Neutrophils
Douglas B. Kuhns, Debra A. Long Priel, Jessica Chu, Kol A. Zarember
This unit describes the isolation of human polymorphonuclear neutrophils (PMN) from blood using dextran sedimentation and Percoll or Ficoll‐Paque density gradients. Assays of neutrophil functions including respiratory burst activation, phagocytosis, and microbial killing are also described. © 2015
Abstract | Full Text: HTML PDF
The Isolation and Characterization of Murine Macrophages
Ricardo Gonçalves, David M. Mosser
Macrophages are mononuclear phagocytes that are widely distributed throughout the body. These cells can contribute to development and homeostasis and participate in innate and adaptive immune responses. The physiology of macrophages can vary tremendously depending on the environment in which they
Abstract | Full Text: HTML PDF
Activation of Murine Macrophages
David M. Mosser, Ricardo Gonçalves
Our understanding of cell‐mediated immunity (CMI) has revealed the importance of activated macrophages as key immune effector cells. Over the past decade, we have come to realize that macrophages exhibit remarkable plasticity, and different populations of macrophages with distinct physiologies can
Abstract | Full Text: HTML PDF
Trypan Blue Exclusion Test of Cell Viability
Warren Strober
The protocol described in this appendix allows for light microscopic quantitation of cell viability. Cells are suspended in PBS containing trypan blue and then examined to determine the percentage of cells that have clear cytoplasm (viable cells) versus cells that have blue cytoplasm (nonviable
Abstract | Full Text: HTML PDF
Biosafety Oversight and Compliance: What do you Mean, I have to Fill Out Another Form?!
Brenda L. Petrella
This unit is an overview of biosafety compliance and oversight in the United States. Specific attention is given to the oversight of the Institutional Biosafety Committee (IBC) and how the purview of the IBC may overlap with other local committees, such as the Institutional Animal Care and Use
Abstract | Full Text: HTML PDF
Electrotransformation and Clonal Isolation of Rickettsia Species
Sean P. Riley, Kevin R. Macaluso, Juan J. Martinez
Genetic manipulation of obligate intracellular bacteria of the genus Rickettsia is currently undergoing a rapid period of change. The development of viable genetic tools, including replicative plasmids, transposons, homologous recombination, fluorescent protein‐encoding genes, and antibiotic
Abstract | Full Text: HTML PDF
Myxococcus xanthus Growth, Development, and Isolation
Zalman Vaksman, Heidi B. Kaplan
Myxobacteria are a highly social group among the delta proteobacteria that display unique multicellular behaviors during their complex life cycle and provide a rare opportunity to study the boundary between single cells and multicellularity. These organisms are also unusual as their entire life
Abstract | Full Text: HTML PDF
Laboratory Maintenance of Nocardia Species
Dipesh Dhakal, Jae Kyung Sohng
Nocardia spp. are aerobic, Gram‐positive, catalase‐positive, non‐motile actinomycetes. Various species of the genus Nocardia have attracted attention due to their detrimental effects on human health. Recent discoveries, however, have exposed their importance as producers of bioactive compounds and
Abstract | Full Text: HTML PDF
Preparation of Cell Cultures and Vaccinia Virus Stocks
Catherine A. Cotter, Patricia L. Earl, Linda S. Wyatt, Bernard Moss
The culturing of cell lines used with vaccinia virus, both as monolayer and in suspension, is described. The preparation of chick embryo fibroblasts (CEF) is presented for use in the production of the highly attenuated and host range‐restricted modified vaccinia virus Ankara (MVA) strain of vaccinia
Abstract | Full Text: HTML PDF
Generation of Recombinant Vaccinia Viruses
Linda S. Wyatt, Patricia L. Earl, Bernard Moss
This unit describes how to infect cells with vaccinia virus and then transfect them with a plasmid‐transfer vector or PCR fragment to generate a recombinant virus. Selection and screening methods used to isolate recombinant viruses and a method for the amplification of recombinant viruses are
Abstract | Full Text: HTML PDF
Electroblotting from Polyacrylamide Gels
Aaron Goldman, Jeanine A. Ursitti, Jacek Mozdzanowski, David W. Speicher
Transferring proteins from polyacrylamide gels onto retentive membranes is now primarily used for immunoblotting. A second application that was quite common up to about a decade ago was electroblotting of proteins for N‐terminal and internal sequencing using Edman chemistry. This unit contains
Abstract | Full Text: HTML PDF
Electron Microscopy and Image Processing: Essential Tools for Structural Analysis of Macromolecules
David M. Belnap
Macromolecular electron microscopy typically depicts the structures of macromolecular complexes ranging from ∼200 kDa to hundreds of MDa. The amount of specimen required, a few micrograms, is typically 100 to 1000 times less than needed for X‐ray crystallography or nuclear magnetic resonance
Abstract | Full Text: HTML PDF
The Isotope‐Coded Affinity Tag Method for Quantitative Protein Profile Comparison and Relative Quantitation of Cysteine Redox Modifications
James Chun Yip Chan, Lei Zhou, Eric Chun Yong Chan
The isotope‐coded affinity tag (ICAT) technique has been applied to measure pairwise changes in protein expression through differential stable isotopic labeling of proteins or peptides followed by identification and quantification using a mass spectrometer. Changes in protein expression are observed
Abstract | Full Text: HTML PDF
Large‐Scale Identification of the Arginine Methylome by Mass Spectrometry
Kathrine B. Sylvestersen, Michael L. Nielsen
The attachment of one or more methylation groups to the side chain of arginine residues is a regulatory mechanism for cellular proteins. Recent advances in mass spectrometry−based characterization allow comprehensive identification of arginine methylation sites by peptide‐level enrichment
Abstract | Full Text: HTML PDF
Standardized Cryopreservation of Pluripotent Stem Cells
Rick I. Cohen, Maria L. Thompson, Brian Schryver, Rolf O. Ehrhardt
The successful exploitation of human cells for research, translational, therapeutic, and commercial purposes requires that effective and simple cryopreservation methods be applied for storage in local and master cell banks. Of all the cell types utilized in modern research, human embryonic stem
Abstract | Full Text: HTML PDF
Isolation of Mesenchymal Stem Cells from Amniotic Fluid and Placenta
Christopher D. Graham, Dario O. Fauza
Diverse progenitor cell populations, including mesenchymal, hematopoietic, trophoblastic, and possibly more primitive stem cells can be isolated from the amniotic fluid and the placenta. At least some of the amniotic and placental cells share a common origin, namely the inner cell mass of the
Abstract | Full Text: HTML PDF
Quantifying Epithelial Early Common Progenitors from Long‐Term Primary or Cell Line Sphere Culture
Flora Clément, Helen He Zhu, Wei‐Qiang Gao, Emmanuel Delay, Véronique Maguer‐Satta
Here, a protocol to quantify epithelial early common progenitor/stem cells grown as spheres in non‐adherent culture conditions is described. This protocol is based on the combination of two functional tests: the sphere assay to maintain and enrich early progenitor/stem cells, and the epithelial
Abstract | Full Text: HTML PDF
The Derivation of Primary Human Epicardium‐Derived Cells
Caitlin Clunie‐O'Connor, Anke M. Smits, Charalambos Antoniades, Angela J. Russell, Derek M. Yellon, Marie‐José Goumans, Paul R. Riley
To develop therapeutic strategies for the regeneration of lost heart muscle after myocardial infarction (MI), a source of functional new muscle cells and associated coronary vessels must be identified. The epicardium is a source of several cardiovascular cell types during heart development and is
Abstract | Full Text: HTML PDF
CRISPR‐Mediated Gene Targeting of Human Induced Pluripotent Stem Cells
Susan M. Byrne, George M. Church
CRISPR/Cas9 nuclease systems can create double‐stranded DNA breaks at specific sequences to efficiently and precisely disrupt, excise, mutate, insert, or replace genes. However, human embryonic stem cells and induced pluripotent stem cells (iPSCs) are more difficult to transfect and less resilient
Abstract | Full Text: HTML PDF
Use of Ciliogenesis to Detect Aneugens: The Role of Primary Cilia
Kathyayini V. Divi, Yvona Ward, Miriam C. Poirier, Ofelia A. Olivero
Primary cilia arise from the centrosomes of quiescent or post‐mitotic cells, and serve as sensory organelles that communicate mechanical and chemical stimuli from the environment to the interior of the cell. Cilium formation may, therefore, become a useful end point signaling exposure to genotoxins
Abstract | Full Text: HTML PDF
Immune Cell Phenotyping Using Flow Cytometry
A. Graham Pockley, Gemma A. Foulds, Julie A. Oughton, Nancy I. Kerkvliet, Gabriele Multhoff
Fluorescent immunophenotyping uses fluorescently‐conjugated antibodies to identify, characterize and quantify distinct subpopulations of cells within heterogeneous single‐cell populations, either in the context of tissue (using fluorescence and imaging microscopy) or in a single‐cell suspension
Abstract | Full Text: HTML PDF
Caenorhabditis elegans as a Model for Toxic Effects of Nanoparticles: Lethality, Growth, and Reproduction
Laura L. Maurer, Ian T. Ryde, Xinyu Yang, Joel N. Meyer
The nematode Caenorhabditis elegans is extensively utilized in toxicity studies. C. elegans offers a high degree of homology with higher organisms, and its ease of use and relatively inexpensive maintenance have made it an attractive complement to mammalian and ecotoxicological models. C. elegans
Abstract | Full Text: HTML PDF
Measuring p66Shc Signaling Pathway Activation and Mitochondrial Translocation in Cultured Cells
Mariusz R. Wieckowski, Cláudia M. Deus, Renata Couto, Monika Oparka, Magdalena Lebiedzińska‐Arciszewska, Jerzy Duszyński, Paulo J. Oliveira
The adaptor protein p66Shc links membrane receptors to intracellular signaling pathways, with downstream consequences on mitochondrial metabolism and reactive oxygen species production. Moreover, p66Shc has also been implicated in cancer development, progression, and metastasis. Increased
Abstract | Full Text: HTML PDF
Seahorse Xfe24 Extracellular Flux Analyzer‐Based Analysis of Cellular Respiration in Caenorhabditis elegans
Anthony L. Luz, Latasha L. Smith, John P. Rooney, Joel N. Meyer
Mitochondria are critical for their role in ATP production as well as multiple nonenergetic functions, and mitochondrial dysfunction is causal in myriad human diseases. Less well appreciated is the fact that mitochondria integrate environmental and intercellular as well as intracellular signals to
Abstract | Full Text: HTML PDF