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Latest Articles

ELISPOT Assay for Measurement of Antigen‐Specific and Polyclonal Antibody Responses
Nils Lycke, Richard Coico

The enzyme‐linked immunospot (ELISPOT) assay for detection of antigen‐specific and polyclonal antibody responses by single antibody‐secreting cells has become the method of choice due to its cell‐based quantitative value.

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Adriamycin Nephropathy in BALB/c Mice
Yuan Min Wang, Yiping Wang, David C. H. Harris, Stephen I. Alexander, Vincent W. S. Lee

Chronic proteinuric renal injury is a major cause of end stage renal disease. Adriamycin nephropathy (AN) is a murine model of chronic proteinuric renal disease whereby chemical injury is followed by immune and structural changes that mimic human disease.

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Animal Models for the Analysis of Immune Responses to Leishmaniasis
David L. Sacks, Peter C. Melby

This unit focuses on the murine model of cutaneous leishmaniasis and models of visceral leishmaniasis in mice and hamsters.

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Mouse Model for Pre‐Clinical Study of Human Cancer Immunotherapy
Zhiya Ya, Yared Hailemichael, Willem Overwijk, Nicholas P. Restifo

This unit describes protocols for developing tumors in mice, including subcutaneous growth, pulmonary metastases of B16 melanoma, and spontaneous melanoma in B‐Raf V600E/PTEN deletion transgenic mouse models. Two immunization methods to prevent B16 tumor growth are described using B16.

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Genome‐Wide Fitness and Genetic Interactions Determined by Tn‐seq, a High‐Throughput Massively Parallel Sequencing Method for Microorganisms
Tim van Opijnen, David W. Lazinski, Andrew Camilli

The lagging annotation of bacterial genomes and the inherent genetic complexity of many phenotypes is hindering the discovery of new drug targets and the development of new antimicrobial agents and vaccines.

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Streptococcus pneumoniae Supragenome Hybridization Arrays for Profiling of Genetic Content and Gene Expression
Anagha Kadam, Benjamin Janto, Rory Eutsey, Joshua P. Earl, Evan Powell, Margaret E. Dahlgren, Fen Z. Hu, Garth D. Ehrlich, N. Luisa Hiller

There is extensive genomic diversity among Streptococcus pneumoniae isolates.

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Aminoglycoside‐Enabled Elucidation of Bacterial Persister Metabolism
Mehmet A. Orman, Wendy W. K. Mok, Mark P. Brynildsen

Bacterial persisters are cells with an impressive, yet transient, tolerance toward extraordinary concentrations of antibiotics. Persisters are believed to impose a significant burden on the healthcare system because of their role in the proclivity of infections to relapse.

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Progress in Vaccine Development
Peter Michael Moyle

Vaccination has a proven record as one of the most effective medical approaches to prevent the spread of infectious diseases. Traditional vaccine approaches involve the administration of whole killed or weakened microorganisms to stimulate protective immune responses.

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Application of meta‐ and para‐Phenylenediamine as Enhanced Oxime Ligation Catalysts for Protein Labeling, PEGylation, Immobilization, and Release
Mohammad M. Mahmoodi, Mohammad Rashidian, Yi Zhang, Mark D. Distefano

Meta‐ and para‐phenylenediamines have recently been shown to catalyze oxime and hydrazone ligation reactions at rates much faster than aniline, a commonly used catalyst.

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Measuring Protein‐Protein and Protein‐Nucleic Acid Interactions by Biolayer Interferometry
Azmiri Sultana, Jeffrey E. Lee

Biolayer interferometry (BLI) is a simple, optical dip‐and‐read system useful for measuring interactions between proteins, peptides, nucleic acids, small molecules, and/or lipids in real time. In BLI, a biomolecular bait is immobilized on a matrix at the tip of a fiber‐optic sensor.

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Analysis of Protein Ligand‐Receptor Binding by Photoaffinity Cross‐Linking
Ling Wu, Bin Xu

Photoaffinity cross‐linking is a rapidly developing technology for studying biomolecular interactions, including protein ligand‐receptor binding.

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Analysis of Protein Stability and Ligand Interactions by Thermal Shift Assay
Kathy Huynh, Carrie L. Partch

Purification of recombinant proteins for biochemical assays and structural studies is time‐consuming and presents inherent difficulties that depend on the optimization of protein stability.

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Ligand‐Directed Profiling of Organelles with Internalizing Phage Libraries
Andrey S. Dobroff, Roberto Rangel, Liliana Guzman‐Roja, Carolina C. Salmeron, Juri G. Gelovani, Richard L. Sidman, Cristian G. Bologa, Tudor I. Oprea, C. Jeffrey Brinker, Renata Pasqualini, Wadih Arap

Phage display is a resourceful tool to, in an unbiased manner, discover and characterize functional protein‐protein interactions, create vaccines, and engineer peptides, antibodies, and other proteins as targeted diagnostic and/or therapeutic agents.

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Efficient and Scalable Culture of Single Dissociated Human Pluripotent Stem Cells Using Recombinant E8 Fragments of Human Laminin Isoforms
Takamichi Miyazaki, Eihachiro Kawase

This unit describes a protocol for efficient expansion of human pluripotent stem cells (hPSCs). A key feature of this method is subculture of hPSCs by single‐cell dissociation passaging on substrates coated with recombinant E8 fragments of human laminin isoforms (LM‐E8s).

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Isolation and Manipulation of Mouse Trophoblast Stem Cells
Koji Hayakawa, Emi Himeno, Satoshi Tanaka, Tilo Kunath

The isolation of stable trophoblast stem (TS) cell lines from early mouse embryos has provided a useful cell culture model to study trophoblast development. TS cells are derived from pre‐implantation blastocysts or from the extraembryonic ectoderm of early post‐implantation embryos.

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Generation of Highly Enriched Populations of Optic Vesicle−Like Retinal Cells from Human Pluripotent Stem Cells
Sarah K. Ohlemacher, Clara L. Iglesias, Akshayalakshmi Sridhar, David M. Gamm, Jason S. Meyer

The protocol outlined below is used to differentiate human pluripotent stem cells (hPSCs) into retinal cell types through a process that faithfully recapitulates the stepwise progression observed in vivo.

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Teratoma Formation: A Tool for Monitoring Pluripotency in Stem Cell Research
Raman V. Nelakanti, Nigel G. Kooreman, Joseph C. Wu

This unit describes protocols for evaluating the pluripotency of embryonic and induced pluripotent stem cells using a teratoma formation assay.

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Metabolism of Xenobiotics by Aldehyde Oxidase
Deepak Dalvie, Michael Zientek

Aldehyde oxidase (AO) is a cytosolic molybdoflavoprotein whose contribution to the metabolism and clearance of xenobiotics‐containing heterocyclic rings has attracted increased interest in recent years.

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Methods to Measure Olfactory Behavior in Mice
Junhui Zou, Wenbin Wang, Yung‐Wei Pan, Song Lu, Zhengui Xia

Mice rely on the sense of olfaction to detect food sources, recognize social and mating partners, and avoid predators.

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How to Measure Alterations in Alveolar Barrier Function as a Marker of Lung Injury
Raquel Herrero, Gustavo Matute‐Bello

The alveolar capillary membrane maintains the proper water and solute content of the epithelial lining fluid at the alveolar air‐liquid interface, which is critical for adequate gas exchange in the lung.

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Inhalation Toxicology Methods: The Generation and Characterization of Exposure Atmospheres and Inhalational Exposures
Lung‐Chi Chen, Morton Lippmann

In this unit, the need for laboratory‐based inhalation toxicology studies, the historical background on adverse health effects of airborne toxicants, and the benefits of advance planning for the building of analytic options into the study design to maximize the scientific gains to be derive.

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