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Collection, Storage, and Preparation of Human Blood Cells
Pradeep K. Dagur, J. Philip McCoy
Human peripheral blood is often studied by flow cytometry in both the research and clinical laboratories. The methods for collection, storage, and preparation of peripheral blood will vary depending on the cell lineage to be examined as well as the type of assay to be performed. This unit presents
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High Throughput‐Based Mitochondrial Function Assays by Multi‐Parametric Flow Cytometry
J. Paul Robinson, Nianyu Li, Padma Kumar Narayanan
Mitochondrial dysfunction has been increasingly implicated as an important mechanism for chemical‐induced toxicity. In the present unit, we describe a multi‐parametric flow cytometry assay to assess the effects of drug or chemical‐induced mitochondrial dysfunction in cells. Cells are cultured in a
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Visualization of Telomere Integrity and Function In Vitro and In Vivo Using Immunofluorescence Techniques
Anthony J. Cesare, Christopher M. Heaphy, Roderick J. O'Sullivan
In cancer cells, telomere length maintenance occurs largely via the direct synthesis of TTAGGG repeats at chromosome ends by telomerase, or less frequently by the recombination‐dependent alternative lengthening of telomeres (ALT) pathway. The latter is characterized by the atypical clustering of
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Flow Cytometry of Extracellular Vesicles: Potential, Pitfalls, and Prospects
John P. Nolan
Evidence suggests that extracellular vesicles (EVs) can play roles in physiology and pathology, providing impetus to explore their use as diagnostic and therapeutic targets. However, EVs are also small, heterogeneous, and difficult to measure, and so this potential has not yet been realized. The
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Using the PhenX Toolkit to Add Standard Measures to a Study
Tabitha Hendershot, Huaqin Pan, Jonathan Haines, William R. Harlan, Mary L. Marazita, Catherine A. McCarty, Erin M. Ramos, Carol M. Hamilton
The PhenX (consensus measures for Phenotypes and eXposures) Toolkit (https://www.phenxtoolkit.org/) offers high‐quality, well‐established measures of phenotypes and exposures for use by the scientific community. The goal is to promote the use of standard measures, enhance data interoperability, and
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Glycosylation Analysis for Congenital Disorders of Glycosylation
Xueli Li, Mohd A. Raihan, Francis Jeshira Reynoso, Miao He
Congenital disorders of glycosylation (CDG) are a group of diseases with highly variable phenotypes and inconsistent clinical features. Since the first description of a CDG in 1980, approximately 100 disorders have been identified. Most of these are defects in protein glycosylation, although an
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Determination of Activity of the Enzymes Hypoxanthine Phosphoribosyl Transferase (HPRT) and Adenine Phosphoribosyl Transferase (APRT) in Blood Spots on Filter Paper
Kasie Auler, Robyn Broock, William L. Nyhan
Hypoxanthine‐guanine phosphoribosyl‐transferase (HPRT) deficiency is the cause of Lesch‐Nyhan disease. Adenine phosphoribosyl‐transferase (APRT) deficiency causes renal calculi. The activity of each enzyme is readily determined on spots of whole blood on filter paper. This unit describes a method
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Porphyria Diagnostics—Part 1: A Brief Overview of the Porphyrias
Vaithamanithi‐Mudumbai Sadagopa Ramanujam, Karl Elmo Anderson
Porphyria diseases are a group of metabolic disorders caused by abnormal functioning of heme biosynthesis enzymes and characterized by excessive accumulation and excretion of porphyrins and their precursors. Precisely which of these chemicals builds up depends on the type of porphyria. Porphyria is
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Finding Pathogenic Nucleic Acid Sequences in Next Generation Sequencing Data
Michael Parfenov, J.G. Seidman
Viruses and bacteria are established as one of the main causes of human diseases from hepatitis to cancer. Recently, the presence of such pathogens has been extensively studied using human whole genome and transcriptome sequencing data. However, detecting and studying pathogens via next generation
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Isolation of Nascent Transcripts with Click Chemistry
Ozlem Yildirim
Steady‐state levels of cellular RNA are determined by both transcriptional rate and RNA half‐life. Commonly used methods for transcriptional analysis are only capable of profiling total RNA and do not distinguish changes in synthesis and decay rates. Hence, a better understanding of the temporal
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Parallel WGA and WTA for Comparative Genome and Transcriptome NGS Analysis Using Tiny Cell Numbers
Christian Korfhage, Evelyn Fricke, Andreas Meier
Genomic DNA determines how and when the transcriptome is changed by a trigger or environmental change and how cellular metabolism is influenced. Comparative genome and transcriptome analysis of the same cell sample links a defined genome with all changes in the bases, structure, or numbers of the
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Whole‐Transcriptome Amplification of Single Cells for Next‐Generation Sequencing
Christian Korfhage, Evelyn Fricke, Andreas Meier
Single‐cell transcriptome analysis gives insight into the heterogeneity of neighboring cells in tissues or cell cultures. It has been shown that the variability of cells is important for biological function. However, cell variability can be an indication of disease (e.g., cancer). In order to
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Using Google Reverse Image Search to Decipher Biological Images
Jennifer L. Mamrosh, David D. Moore
Despite the range of tasks performed by biological image‐processing software, current versions cannot find matches for the image in question among the huge range of biological images that exist in the literature and elsewhere on the Internet. Google's Reverse Image Search is designed for this, and
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MARCC (Matrix‐Assisted Reader Chromatin Capture): An Antibody‐Free Method to Enrich and Analyze Combinatorial Nucleosome Modifications
Zhangli Su, John M. Denu
Combinatorial patterns of histone modifications are key indicators of different chromatin states. Most of the current approaches rely on the usage of antibodies to analyze combinatorial histone modifications. Here we detail an antibody‐free method named MARCC (Matrix‐Assisted Reader Chromatin
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Gene Silencing by RNAi in Mammalian Cells
Frida Ponthan, Narazah Mohd Yusoff, Natalia Martinez Soria, Olaf Heidenreich, Kelly Coffey
This unit provides information how to use short interfering RNA (siRNA) for sequence‐specific gene silencing in mammalian cells. Several methods for siRNA generation and optimization, as well as recommendations for cell transfection and transduction, are presented. © 2015 by John Wiley & Sons,
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Assaying Cell Cycle Status Using Flow Cytometry
Kang Ho Kim, Joel M. Sederstrom
In this unit, two protocols are described for analyzing cell cycle status using flow cytometry. The first is based on the simultaneous analysis of proliferation‐specific marker (Ki‐67) and cellular DNA content, which discriminate resting/quiescent cell populations (G0 cell) and quantify cell cycle
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CRISPR‐Cas9 Genome Editing in Drosophila
Scott J. Gratz, C. Dustin Rubinstein, Melissa M. Harrison, Jill Wildonger, Kate M. O'Connor‐Giles
The CRISPR‐Cas9 system has transformed genome engineering of model organisms from possible to practical. CRISPR‐Cas9 can be readily programmed to generate sequence‐specific double‐strand breaks that disrupt targeted loci when repaired by error‐prone non‐homologous end joining (NHEJ) or to catalyze
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Cre Activated and Inactivated Recombinant Adeno‐Associated Viral Vectors for Neuronal Anatomical Tracing or Activity Manipulation
Arpiar Saunders, Bernardo L. Sabatini
Recombinant adeno‐associated viruses (rAAVs) transcriptionally activated by Cre recombinase (Cre‐On) are powerful tools for determining the anatomy and function of genetically defined neuronal types in transgenic Cre driver mice. Here we describe how rAAVs transcriptionally inactivated by Cre
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Time‐Lapse Imaging of Glial–Axonal Interactions
Kalliopi Ioannidou, Julia M. Edgar, Susan C. Barnett
In the central nervous system (CNS), myelin is formed by oligodendrocytes that are derived from precursor cells, known as oligodendrocyte precursor cells (OPCs). Successive stages of OPC interactions with the axons can be visualized in vitro and ex vivo using mixed neural cell cultures and pieces of
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The Use of Herpes Simplex Virus in Ex Vivo Slice Culture
Allyson K. Friedman, Ming‐Hu Han
Herpes simplex virus (HSV) can be used for a wide range of genetic manipulations in ex vivo slices of central nervous system tissue from both young and adult rodents. The fast expression of the HSV viral‐mediated gene transfer, which can be engineered to produce cell‐type specificity, can be
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Attentional Set‐Shifting Paradigm in the Rat
Piotr Popik, Agnieszka Nikiforuk
The attentional set‐shifting task (ASST) is the rat version of the intra‐dimensional/extra‐dimensional (ID/ED) test and was developed fifteen years ago. Damage to the medial frontal cortex results in a failure to “unlearn” old contingencies, i.e., impairs ED set shifting. As such, the task measures
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