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

Latest Articles

High‐Dimensional Single‐Cell Analysis with Mass Cytometry
Tess Melinda Brodie, Vinko Tosevski
Mass cytometry is an analytical technology that combines the sample preparation workflow typical of flow cytometry and the detection capacity of atomic mass spectroscopy, allowing for highly multiplexed measurements of protein or nucleic acid targets on single cells. In 2014, the mass cytometer was
Abstract | Full Text: HTML PDF
A Real‐Time Cytotoxicity Assay as an Alternative to the Standard Chromium‐51 Release Assay for Measurement of Human NK and T Cell Cytotoxic Activity
Julien Fassy, Kyriaki Tsalkitzi, Maria Goncalves‐Maia, Véronique M. Braud
This unit describes the monitoring and quantification of cellular cytotoxicity using a non‐radioactive and real‐time cytotoxic assay. The extent of target‐cell lysis is monitored over time by imaging and quantifying live fluorescent target cells using a cell‐imaging multimode reader. This assay is
Abstract | Full Text: HTML PDF
Characterization of Human Blood Monocytes and Intestinal Macrophages
Evida A. Dennis, Tanya O. Robinson, Lesley E. Smythies, Phillip D. Smith
Monocytes and macrophages play fundamental roles in defense against microbes, clearance of senescent and dead cells, and immunoregulation. Although blood monocytes are the source of intestinal macrophages in the developed mucosal immune system, blood monocytes and intestinal macrophages from healthy
Abstract | Full Text: HTML PDF
Nomenclature and Serology of HLA Class I and Class II Alleles
James McCluskey, Carmel Kanaan, Mary Diviney
This overview presents nomenclature and serology information on human leucocyte antigens, or HLA molecules, which are encoded by a cluster of genes linked on the short arm of chromosome 6. This region is known as the major histocompatibility complex and codes for class I and class II molecules,
Abstract | Full Text: HTML PDF
Venipuncture Procedures: Sources of Human Peripheral Blood Cells, Serum, and Plasma
Barbara E. Bierer, Warren Strober
Peripheral blood is the chief source of mononuclear cells (lymphocytes and monocytes) for immunologic studies of humans. This appendix presents protocols for obtaining blood by simple venipuncture when relatively small amounts of blood (10 to 100 ml) are necessary or by lymphapheresis when large
Abstract | Full Text: HTML PDF
Universal Precautions: Necessary Safety Procedures When Handling Human Blood, Body Fluids, and Specimens
Barbara E. Bierer
Universal precautions are observed whenever handling human blood, body fluids, or specimens as a means of preventing exposure to blood‐borne pathogens. This appendix outlines safety procedures to follow whenever undertaking research activities that involve human blood, body fluids, and specimens. ©
Abstract | Full Text: HTML PDF
Using KBase to Assemble and Annotate Prokaryotic Genomes
Benjamin Allen, Meghan Drake, Nomi Harris, Tarah Sullivan
The DOE Systems Biology Knowledgebase (KBase, http://kbase.us/) is an open‐access bioinformatics software and data platform for analyzing plants, microbes, and their communities. KBase enables scientists to create, execute, collaborate on, and share reproducible analyses of their biological data in
Abstract | Full Text: HTML PDF
Quantitative Dextran Trafficking to the Coxiella burnetii Parasitophorous Vacuole
Seth Winfree, Stacey D. Gilk
The gram‐negative bacterium Coxiella burnetii causes human Q fever, a disease characterized by a debilitating flu‐like illness in acute cases and endocarditis in chronic patients. An obligate intracellular pathogen, Coxiella burnetii survives within a large, lysosome‐like vacuole inside the host
Abstract | Full Text: HTML PDF
Mouse Models of Acinetobacter baumannii Infection
Greg Harris, Rhonda KuoLee, H. Howard Xu, Wangxue Chen
This unit describes basic protocols for infecting mice through intranasal and intraperitoneal routes with Acinetobacter baumannii to induce associated pneumonia and sepsis, the two most common manifestations of clinical infections with this pathogen. By selecting the appropriate protocols and
Abstract | Full Text: HTML PDF
Epigenetic Analysis of SV40 Minichromosomes
Lata Balakrishnan, Barry Milavetz
Simian virus 40 (SV40) is one of the best‐characterized members of the polyomavirus family of small DNA tumor viruses. It has a small genome of 5243 bp and utilizes cellular proteins for its molecular biology, with the exception of the T‐antigen protein, which is coded by the virus and is involved
Abstract | Full Text: HTML PDF
Generating and Maintaining Transgenic Cryptosporidium parvum Parasites
Mattie C. Pawlowic, Sumiti Vinayak, Adam Sateriale, Carrie F. Brooks, Boris Striepen
The apicomplexan parasite Cryptosporidium is a leading cause of diarrheal disease and an important contributor to overall global child mortality. We currently lack effective treatment and immune prophylaxis. Recent advances now permit genetic modification of this important pathogen. We expect this
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
Purification of Recombinant Human Tyrosinase from Insect Larvae Infected with the Baculovirus Vector
Monika B. Dolinska, Paul T. Wingfield, Yuri V. Sergeev
The purification of an enzyme from insect larvae infected with a baculovirus vector is described. The enzyme tyrosinase is of biomedical importance and catalyzes the first rate‐limiting steps in melanin production. Tyrosinase mutations can result in oculocutaneous albinism type 1 (OCA1), an
Abstract | Full Text: HTML PDF
Mass‐Tag Labeling Using Acyl‐PEG Exchange for the Determination of Endogenous Protein S‐Fatty Acylation
Avital Percher, Emmanuelle Thinon, Howard Hang
The covalent coupling of fatty acids to proteins provides an important mechanism of regulation in cells. In eukaryotes, cysteine fatty acylation (S‐fatty acylation) has been shown to be critical for protein function in a variety of cellular pathways as well as microbial pathogenesis. While methods
Abstract | Full Text: HTML PDF
Site‐Specific Protein Labeling via Sortase‐Mediated Transpeptidation
John M. Antos, Jessica Ingram, Tao Fang, Novalia Pishesha, Matthias C. Truttmann, Hidde L. Ploegh
Strategies for site‐specific protein modification are highly desirable for the construction of conjugates containing non‐genetically‐encoded functional groups. Ideally, these strategies should proceed under mild conditions, and be compatible with a wide range of protein targets and non‐natural
Abstract | Full Text: HTML PDF
Protein‐Lipid Interaction by Fluorescence (PLIF) to Characterize and Screen for Inhibitors of Protein‐Phosphoinositide Interactions
Laurie Ceccato, Mélanie Mansat, Bernard Payrastre, Frédérique Gaits‐Iacovoni, Julien Viaud
Phosphoinositides are key signaling and regulatory phospholipids that mediate important pathophysiological processes. This is achieved through the interaction of their phosphorylated inositol head group with a wide range of protein domains. Therefore, being able to determine the phosphoinositide
Abstract | Full Text: HTML PDF
Functional Stem Cell Integration into Neural Networks Assessed by Organotypic Slice Cultures
David Forsberg, Charoensri Thonabulsombat, Johan Jäderstad, Linda Maria Jäderstad, Petri Olivius, Eric Herlenius
Re‐formation or preservation of functional, electrically active neural networks has been proffered as one of the goals of stem cell–mediated neural therapeutics. A primary issue for a cell therapy approach is the formation of functional contacts between the implanted cells and the host tissue.
Abstract | Full Text: HTML PDF
Generation of Oligodendrogenic Spinal Neural Progenitor Cells From Human Induced Pluripotent Stem Cells
Mohamad Khazaei, Christopher S. Ahuja, Michael G. Fehlings
This unit describes protocols for the efficient generation of oligodendrogenic neural progenitor cells (o‐NPCs) from human induced pluripotent stem cells (hiPSCs). Specifically, detailed methods are provided for the maintenance and differentiation of hiPSCs, human induced pluripotent stem
Abstract | Full Text: HTML PDF
Generation of Xeno‐Free, cGMP‐Compliant Patient‐Specific iPSCs from Skin Biopsy
Luke A. Wiley, Kristin R. Anfinson, Cathryn M. Cranston, Emily E. Kaalberg, Malia M. Collins, Robert F. Mullins, Edwin M. Stone, Budd A. Tucker
This unit describes protocols for the generation of clinical‐grade patient‐specific induced pluripotent stem cell (iPSC)–derived retinal cells from patients with inherited retinal degenerative blindness. Specifically, we describe how, using xeno‐free reagents in an ISO class 5 environment, one can
Abstract | Full Text: HTML PDF
Development of Hepatocyte‐like Cell Derived from Human Induced Pluripotent Stem cell as a Host for Clinically Isolated Hepatitis C Virus
Khanit Sa‐ngiamsuntorn, Suradej Hongeng, Adisak Wongkajornsilp
This unit describes protocols to develop hepatocyte‐like cells (HLCs) starting from mesenchymal stem cells (MSCs) as a natural host for hepatitis C virus (HCV). These include the preparation of MSCs from bone marrow, the reprogramming of MSCs into induced pluripotent stem cells (iPSCs), and the
Abstract | Full Text: HTML PDF
Production of Single Contracting Human Induced Pluripotent Stem Cell‐Derived Cardiomyocytes: Matrigel Mattress Technique
Adrian G. Cadar, Tromondae K. Feaster, Matthew D. Durbin, Charles C. Hong
This unit describes the published Matrigel mattress method. Briefly, we describe the preparation of the mattress, replating of the human induced pluripotent stem cell–derived cardiomyocytes (hiPSC‐CM) on the Matrigel mattress, and hiPSC‐CM mattress maintenance. Adherence to this protocol will yield
Abstract | Full Text: HTML PDF
Direct Cell Labeling to Image Transplanted Stem Cells in Real Time Using a Dual‐Contrast MRI Technique
Ethel J. Ngen, Yoshinori Kato, Dmitri Artemov
Exogenous direct cell labeling with superparamagnetic iron oxide nanoparticles (SPIONs) is currently the most employed cell‐labeling technique for tracking transplanted cells using magnetic resonance imaging (MRI). Although SPION‐based cell labeling is effective for monitoring cell delivery and
Abstract | Full Text: HTML PDF
3D Differentiation of LUHMES Cell Line to Study Recovery and Delayed Neurotoxic Effects
Georgina Harris, Helena Hogberg, Thomas Hartung, Lena Smirnova
Current neurotoxicity testing and the study of molecular mechanisms in neurodegeneration in vitro usually focuses on acute exposures to compounds. 3D Lund human mesencephalic (LUHMES) cells allow long‐term treatment or pulse exposure in combination with compound washout to study delayed neurotoxic
Abstract | Full Text: HTML PDF
Microphysiological Systems to Assess Nonclinical Toxicity
Kirk P. Van Ness, Shih‐Yu Chang, Elijah J. Weber, Danielle Zumpano, David L. Eaton, Edward J. Kelly
The liver and the kidney are key toxicity target organs during drug development campaigns, as they typically carry the burden of drug transport and metabolism. Primary hepatocytes and proximal tubule epithelial cells grown in traditional in vitro 2‐D culture systems do not maintain transporter and
Abstract | Full Text: HTML PDF
Intracellular Cytokine Detection by Flow Cytometry in Surface Marker‐Defined Human Peripheral Blood Mononuclear T Cells
Fredine T. Lauer, Jesse L. Denson, Ellen Beswick, Scott W. Burchiel
In a recent unit in this series, protocols for the isolation, cryopreservation, thawing, and immunophenotyping of HPBMC isolated from peripheral whole blood using cell surface marker (CSM) staining and multi‐color flow cytometry analysis were presented. The current procedure describes the detection
Abstract | Full Text: HTML PDF
Combining Cytotoxicity Assessment and Xenopus laevis Phenotypic Abnormality Assay as a Predictor of Nanomaterial Safety
Karamallah Al‐Yousuf, Carl A. Webster, Grant N. Wheeler, Francesca Baldelli Bombelli, Victoria Sherwood
The African clawed frog, Xenopus laevis , has been used as an efficient pre‐clinical screening tool to predict drug safety during the early stages of the drug discovery process. X. laevis is a relatively inexpensive model that can be used in whole organism high‐throughput assays whilst maintaining a
Abstract | Full Text: HTML PDF
Optimizing Dual Fluorescent Analysis to Investigate the Toxicity of AgNPs in E. coli
Wei Hong, Shaopeng Chen
The ever‐increasing use of silver nanoparticles (AgNPs) carries potential ecotoxicological risks. For full risk assessment, E. coli cells harboring a plasmid with a constitutively expressed GFP gene under control of lac promoter ( lac::GFP ) are extensively utilized. Flow cytometry is an
Abstract | Full Text: HTML PDF
Use of Human Pluripotent Stem Cell Derived‐Cardiomyocytes to Study Drug‐Induced Cardiotoxicity
Agnes Maillet, Kim Peng Tan, Liam R. Brunham
Drug‐induced cardiotoxicity is the one of the most common causes of drug withdrawal from market. A major barrier in managing the risk of drug‐induced cardiotoxicity has been the lack of relevant models to study cardiac safety. Human pluripotent stem cell‐derived cardiomyocytes (hPSC‐CMs) have great
Abstract | Full Text: HTML PDF