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Lasers for Flow Cytometry: Current and Future Trends
Howard M. Shapiro, William G. Telford
Lasers are the principal light sources for flow cytometers. Virtually all cytometers are equipped with at least one (and often many more) lasers. This unit covers the various types of lasers available and the qualities that make them suitable or unsuitable for use in flow cytometers. Also included
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Non‐Parametric Comparison of Single Parameter Histograms
James C.S. Wood
A number of methods have been developed to compare single parameter histograms. Some perform a channel‐by‐channel analysis and others give a single statistic about how the histograms may or may not differ. If they do differ, then the significance of the difference or confidence limit is usually
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Generating Quantitative Cell Identity Labels with Marker Enrichment Modeling (MEM)
Kirsten E. Diggins, Jocelyn S. Gandelman, Caroline E. Roe, Jonathan M. Irish
Multiplexed single‐cell experimental techniques like mass cytometry measure 40 or more features and enable deep characterization of well‐known and novel cell populations. However, traditional data analysis techniques rely extensively on human experts or prior knowledge, and novel machine learning
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Basics of Digital Microscopy
Callen T. Wallace, Morgan Jessup, Tytus Bernas, Karina A. Peña, Michael J. Calderon, Patricia A. Loughran
Modern digital microscopy combines the equipment of classical light microscopy with a computerized imaging system. The technique comprises image formation by optics, image registration by a camera, and saving of image data in a computer file. This chapter describes limitations that are particular to
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Live‐Animal Imaging of Renal Function by Multiphoton Microscopy
Kenneth W. Dunn, Timothy A. Sutton, Ruben M. Sandoval
Intravital microscopy, microscopy of living animals, is a powerful research technique that combines the resolution and sensitivity found in microscopic studies of cultured cells with the relevance and systemic influences of cells in the context of the intact animal. The power of intravital
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Balanced Chromosomal Rearrangement Detection by Low‐Pass Whole‐Genome Sequencing
Zirui Dong, Lingfei Ye, Zhenjun Yang, Haixiao Chen, Jianying Yuan, Huilin Wang, Xiaosen Guo, Yun Li, Jun Wang, Fang Chen, Sau Wai Cheung, Cynthia C. Morton, Hui Jiang, Kwong Wai Choy
Balanced chromosomal rearrangements (or balanced chromosome abnormalities, BCAs) are common chromosomal structural variants. Emerging studies have demonstrated the feasibility of using whole‐genome sequencing (WGS) for detection of BCA‐associated breakpoints, but the requirement for a priori
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Biosafety in Handling Gene Transfer Vectors
Scott Swindle
This unit is devoted to safety issues that must be considered when generating and working with the most common vectors under development for human gene therapy today. © 2018 by John Wiley & Sons, Inc. Keywords: gene transfer; vectors; biosafety
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CRISPR/Cas9‐Mediated Fluorescent Tagging of Endogenous Proteins in Human Pluripotent Stem Cells
Arun Sharma, Christopher N. Toepfer, Tarsha Ward, Lauren Wasson, Radhika Agarwal, David A. Conner, Johnny H. Hu, Christine E. Seidman
Human induced pluripotent stem cells (hiPSCs) can be used to mass produce surrogates of human tissues, enabling new advances in drug screening, disease modeling, and cell therapy. Recent developments in clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 genome editing technology
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Differentiation and Contractile Analysis of GFP‐Sarcomere Reporter hiPSC‐Cardiomyocytes
Arun Sharma, Christopher N. Toepfer, Manuel Schmid, Amanda C. Garfinkel, Christine E. Seidman
Human induced pluripotent stem cell‐derived cardiomyocytes (hiPSC‐CMs) represent a powerful cellular platform for illuminating mechanisms of human cardiovascular disease and for pharmacological screening. Recent advances in CRISPR/Cas9‐mediated genome editing technology underlie this profound
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Transfection by Electroporation
Huntington Potter, Richard Heller
Electroporation—the use of high‐voltage electric shocks to introduce DNA into cells—can be used with most cell types, yields a high frequency of both stable transformation and transient gene expression, and, because it requires fewer steps, can be easier than alternate techniques. This unit
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Making Use of Cancer Genomic Databases
Chad J. Creighton
The vast amounts of genomic data now deposited in public repositories represent rich resources for cancer researchers. Large‐scale genomics initiatives such as The Cancer Genome Atlas have made available data from multiple molecular profiling platforms (e.g., somatic mutation, RNA and protein
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Avian Retrovirus‐Mediated Tumor‐Specific Gene Knockout
Wei Wang, Bingning Dong, Feng Yang
The RCAS (replication‐competent avian sarcoma leukosis virus long‐terminal repeat with splice acceptor)‐TVA (tumor virus A) gene delivery system has been successfully used in modeling human cancers. Based on this, we have recently developed a novel RCI‐Oncogene (RCAS‐Cre‐IRES‐Oncogene) gene delivery
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CRISPR/Cas9‐Mediated Genome Editing in Epstein‐Barr Virus‐Transformed Lymphoblastoid B‐Cell Lines
Sizun Jiang, Liang Wei Wang, Michael J. Walsh, Stephen J. Trudeau, Catherine Gerdt, Bo Zhao, Benjamin E. Gewurz
Epstein‐Barr virus (EBV) efficiently transforms primary human B cells into immortalized lymphoblastoid cell lines (LCLs), which are extensively used in human genetic, immunological and virological studies. LCLs provide unlimited sources of DNA for genetic investigation, but can be difficult to
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Modulating Gene Expression in Epstein‐Barr Virus (EBV)‐Positive B Cell Lines with CRISPRa and CRISPRi
Liang Wei Wang, Stephen J. Trudeau, Chong Wang, Catherine Gerdt, Sizun Jiang, Bo Zhao, Benjamin E. Gewurz
Epstein‐Barr virus (EBV) transforms small resting primary B cells into large lymphoblastoid cells which are able to grow and survive in vitro indefinitely. These cells represent a model for oncogenesis. In this unit, variants of conventional clustered regularly interspaced short palindromic repeats
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CRISPR‐Cas9‐Edited Site Sequencing (CRES‐Seq): An Efficient and High‐Throughput Method for the Selection of CRISPR‐Cas9‐Edited Clones
Yaligara Veeranagouda, Delphine Debono‐Lagneaux, Hamida Fournet, Gilbert Thill, Michel Didier
The emergence of clustered regularly interspaced short palindromic repeats–Cas9 (CRISPR‐Cas9) gene editing systems has enabled the creation of specific mutants at low cost, in a short time and with high efficiency, in eukaryotic cells. Since a CRISPR‐Cas9 system typically creates an array of
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Pooled Lentiviral‐Delivery Genetic Screens
Federica Piccioni, Scott T. Younger, David E. Root
Pooled cell‐based screens of mammalian genetic perturbations enable systematic large‐scale, even genome‐scale, evaluation of gene function. Pooled screens introduce genetic perturbations into a cell population through viral transduction such that each cell integrates into its DNA a single or small
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Hybridization Histochemistry of Neural Transcripts
W. Scott Young, June Song, Éva Mezey
This unit presents protocols to locate RNA transcripts in tissues. Numerous approaches are detailed, including those that use radiolabeled or colorimetric probes. Also, the probes may be modified oligodeoxynucleotides, singly or in pairs, as well as ribonucleic acids. High sensitivity and
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Imaging of Mitochondrial and Cytosolic Ca2+ Signals in Cultured Astrocytes
Nannan Zhang, Shinghua Ding
This unit provides a step‐by‐step protocol for constructing cell type‐ and mitochondria‐targeted GCaMP genetically encoded Ca 2+ indicators (GECIs) for mitochondrial Ca 2+ imaging in astrocytes. Mitochondrial Ca 2+ plays a critical role in controlling cytosolic Ca 2+ buffering, energy metabolism,
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A Guide to Robust Statistical Methods in Neuroscience
Rand R. Wilcox, Guillaume A. Rousselet
There is a vast array of new and improved methods for comparing groups and studying associations that offer the potential for substantially increasing power, providing improved control over the probability of a Type I error, and yielding a deeper and more nuanced understanding of data. These new
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