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High‐Sensitivity Detection of PNH Red Blood Cells, Red Cell Precursors, and White Blood Cells
D. Robert Sutherland, Andrea Illingworth, Michael Keeney, Stephen J. Richards

Flow cytometry is the method of choice to ‘diagnose’ paroxysmal nocturnal hemoglobinuria (PNH) and has led to improved patient management. Most laboratories have limited experience with PNH testing, and many different flow approaches are used.

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Uncompensated Polychromatic Analysis of Mitochondrial Membrane Potential Using JC‐1 and Multilaser Excitation
Sara De Biasi, Lara Gibellini, Andrea Cossarizza

The lipophilic cation JC‐1 (5,5′,6,6′‐tetrachloro‐1,1′,3,3′‐tetraethyl‐benzimidazolyl carbocyanine iodide) has been used for more than 20 years as a specific dye for measuring mitochondrial membrane potential (ΔΨm).

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Cell Proliferation Method: Click Chemistry Based on BrdU Coupling for Multiplex Antibody Staining
Paolo Cappella, Fabio Gasparri, Maurizio Pulici, Jürgen Moll

Determination of incorporation of the thymidine analog 5‐bromo‐2′‐deoxyuridine (BrdU) into DNA is a widely used method to analyze the cell cycle.

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Flow Cytometry of Murine Spermatocytes
Valeriya Gaysinskaya, Alex Bortvin

Protocols for purification of murine male germ cells by FACS based on Hoechst 33342 (Ho342) dye staining have been reported and optimized.

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Measurement of Intracellular Ions by Flow Cytometry
Avery D. Posey,, Omkar U. Kawalekar, Carl H. June

Using flow cytometry, single‐cell measurements of calcium can be made on isolated populations identified by one or more phenotypic characteristics.

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Cell Volume Measurements by Optical Transmission Microscopy
Michael A. Model

Cell volume is an important parameter in cell adaptation to anisosmotic stress, in the development of apoptosis and necrosis, and in the pathogenesis of several diseases. This unit describes a method for measuring the volume of adherent cells using a standard light microscope.

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Diagnosis of Fanconi Anemia by Diepoxybutane Analysis
Arleen D. Auerbach

Fanconi anemia (FA) is a genetically and phenotypically heterogeneous disorder characterized by congenital malformations, progressive bone marrow failure, and predisposition to cancer, particularly hematological malignancies and solid tumors of the head and neck.

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Preparation of Amniocytes for Interphase Fluorescence In Situ Hybridization (FISH)
Stuart Schwartz

FISH has been used to detect and clarify deletions and/or other structural rearrangements, and also has applications in interphase analysis. This unit describes preparation of uncultured amniotic fluid cells for FISH analysis.

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Use of Affymetrix Arrays in the Diagnosis of Gene Copy‐Number Variation
Farah R. Zahir, Marco A. Marra

Diagnosing constitutional pathogenic copy number variants (CNVs) requires detecting submicroscopic segmental chromosomal imbalances. The Affymetrix GeneChip mapping array was one of the initial microarray platforms used to measure duplication and deletion of genetic material in DNA samples.

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Probe‐Directed Degradation (PDD) for Flexible Removal of Unwanted cDNA Sequences from RNA‐Seq Libraries
Stuart K. Archer, Nikolay E. Shirokikh, Thomas Preiss

Most applications for RNA‐seq require the depletion of abundant transcripts to gain greater coverage of the underlying transcriptome. The sequences to be targeted for depletion depend on application and species and in many cases may not be supported by commercial depletion kits.

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Neural Stem Cell‐Mediated Delivery of Oncolytic Adenovirus
Julius W. Kim, J. Robert Kane, Jacob S. Young, Alan L. Chang, Deepak Kanojia, Shuo Qian, Drew A. Spencer, Atique U. Ahmed, Maciej S. Lesniak

The use of stem cells (SCs) as carriers for therapeutic agents has now progressed to early clinical trials.

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Use of Proteasome Inhibitors
Sondra L. Downey, Bogdan I. Florea, Herman S. Overkleeft, Alexei F. Kisselev

Proteasome inhibitors are indispensable research tools in immunology and cell biology. With numerous proteasome inhibitors available commercially, choosing the appropriate compound for a biological experiment may be challenging, especially for a novice.

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Measurement of Bacterial Ingestion and Killing by Macrophages
Douglas A. Drevets, Beth P. Canono, Priscilla A. Campbell

This unit presents assays that allow accurate measurement of phagocytosis and killing of bacteria by macrophages. The first basic protocol describes how to measure the ability of macrophages to ingest bacteria.

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Toll‐Like Receptors
Kiyoshi Takeda, Shizuo Akira

The mammalian Toll‐like receptor (TLR) family consists of 13 members, and recognizes specific patterns of microbial components, called pathogen‐associated molecular patterns (PAMPs).

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Heymann Nephritis in Lewis Rats
Yuan Min Wang, Vincent W.S. Lee, Huiling Wu, David C.H. Harris, Stephen I. Alexander

Human membranous nephritis is a major cause of end‐stage kidney disease. Active Heymann nephritis (HN) is an auto‐immune model of membranous nephritis induced in Lewis rats by immunization with a crude renal tubular antigen (Fx1A) or megalin (gp330).

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Site‐Specific Recombinational Cloning Using Gateway and In‐Fusion Cloning Schemes
Jaehong Park, Andrea L. Throop, Joshua LaBaer

The comprehensive study of protein structure and function, or proteomics, depends on the obtainability of full‐length cDNAs in species‐specific expression vectors and subsequent functional analysis of the expressed protein.

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Mapping 3′ mRNA Isoforms on a Genomic Scale
Yi Jin, Joseph V. Geisberg, Zarmik Moqtaderi, Zhe Ji, Mainul Hoque, Bin Tian, Kevin Struhl

Most eukaryotic genes are transcribed into mRNAs with alternative poly(A) sites. Emerging evidence suggests that mRNA isoforms with alternative poly(A) sites can perform critical regulatory functions in numerous biological processes.

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Purification of Recombinant Proteins from Cultured Mammalian Cells by HaloTag Technology
Rachel Friedman Ohana, Robin Hurst

Cultured mammalian cells provide an environment ideal for producing functional recombinant mammalian proteins. However, low expression levels of recombinant proteins present a challenge for their detection and purification.

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Tools and Procedures for Visualization of Proteins and Other Biomolecules
Lurong Pan, Stephen G. Aller

Protein, peptides, and nucleic acids are biomolecules that drive biological processes in living organisms.

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Mapping Regulatory Factors by Immunoprecipitation from Native Chromatin
Guillermo A. Orsi, Sivakanthan Kasinathan, Gabriel E. Zentner, Steven Henikoff, Kami Ahmad

Occupied Regions of Genomes from Affinity‐purified Naturally Isolated Chromatin (ORGANIC) is a high‐resolution method that can be used to quantitatively map protein‐DNA interactions with high specificity and sensitivity.

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Fixation and Immunolabeling of Brain Slices: SNAPSHOT Method
Lasse Dissing‐Olesen, Brian A. MacVicar

Acute brain slices are widely used in neuroscience because this preparation enables pharmacological interventions in a timely manner, similar to what is currently done in cultured cell studies, while preserving the natural cytoarchitecture.

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Tracking Neuronal Migration in Adult Brain Slices
Karen Bakhshetyan, Armen Saghatelyan

Neuronal migration is one of the fundamental processes underlying the proper assembly and function of neural circuitry. The majority of neuronal precursors are generated far away from their sites of integration and need to migrate substantial distances to reach their final destination.

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Latent Sensitization: A Model for Stress‐Sensitive Chronic Pain
Juan Carlos Marvizon, Wendy Walwyn, Ani Minasyan, Wenling Chen, Bradley K. Taylor

Latent sensitization is a rodent model of chronic pain that reproduces both its episodic nature and its sensitivity to stress. It is triggered by a wide variety of injuries ranging from injection of inflammatory agents to nerve damage.

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Overview of the Purification of Recombinant Proteins
Paul T. Wingfield

When the first version of this unit was written in 1995, protein purification of recombinant proteins was based on a variety of standard chromatographic methods and approaches, many of which were described and mentioned throughout Current Protocols in Protein Science.

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Protein Purification Using PDZ Affinity Chromatography
Ward G. Walkup, Mary B. Kennedy

PDZ domains function in nature as protein‐binding domains within scaffold and membrane‐associated proteins. They comprise approximately 90 residues and undergo specific, high‐affinity interactions with complementary C‐terminal peptide sequences, other PDZ domains, and/or phospholipids.

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Detection of Tyrosine Sulfation on Proteins
Yogita Kanan, Muayyad R. Al Ubaidi

Tyrosine sulfation is a post‐translational modification (PTM) where a sulfate group is added to a tyrosine moiety. This PTM is responsible for strengthening interaction between proteins.

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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.

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Preparing Protein Extracts for Quantitative Two‐Dimensional Gel Comparison
Cécile Lelong, Thierry Rabilloud

This unit describes basic protocols for efficient and reproducible protein solubilization from a variety of biological samples, including cultured animal cells and tissues, plant cells and tissues, bacteria, nuclei, other subcellular organelles, plasma, serum, and other biological fluids.

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