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Current Protocols in Cytometry

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https://www.readbyqxmd.com/read/29345330/non-parametric-comparison-of-single-parameter-histograms
#1
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 provided. The specific location(s) for the greatest deviations may also be given. Some are more effective at resolving severely overlapping populations and others work poorly when there is any significant overlap. Each method makes certain assumptions about the data...
January 18, 2018: Current Protocols in Cytometry
https://www.readbyqxmd.com/read/29345329/generating-quantitative-cell-identity-labels-with-marker-enrichment-modeling-mem
#2
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 algorithms may generate unexpected population groupings. Marker enrichment modeling (MEM) creates quantitative identity labels based on features enriched in a population relative to a reference. While developed for cell type analysis, MEM labels can be generated for a wide range of multidimensional data types, and MEM works effectively with output from expert analysis and diverse machine learning algorithms...
January 18, 2018: Current Protocols in Cytometry
https://www.readbyqxmd.com/read/29345328/lasers-for-flow-cytometry-current-and-future-trends
#3
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 is a discussion of future directions, particularly in the area of tunable laser development. Practical tips are provided for building multilaser cytometer systems. © 2018 by John Wiley & Sons, Inc.
January 18, 2018: Current Protocols in Cytometry
https://www.readbyqxmd.com/read/29345327/basics-of-digital-microscopy
#4
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 each of these processes, including optical resolution, efficiency of image registration, characteristics of image file formats, and data management. Further suggestions are given which serve, in turn, to help construct a set of guidelines aimed at optimization of digital microscopic imaging...
January 18, 2018: Current Protocols in Cytometry
https://www.readbyqxmd.com/read/29345326/live-animal-imaging-of-renal-function-by-multiphoton-microscopy
#5
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 microscopy has recently been extended with the development of multiphoton fluorescence microscopy systems capable of collecting optical sections from deep within the kidney at subcellular resolution, supporting high-resolution characterizations of the structure and function of glomeruli, tubules, and vasculature in the living kidney...
January 18, 2018: Current Protocols in Cytometry
https://www.readbyqxmd.com/read/28967991/analysis-of-cellular-dna-content-by-flow-cytometry
#6
Zbigniew Darzynkiewicz, Xuan Huang, Hong Zhao
Cellular DNA content can be measured by flow cytometry with the aim of : (1) revealing cell distribution within the major phases of the cell cycle, (2) estimating frequency of apoptotic cells with fractional DNA content, and/or (3) disclosing DNA ploidy of the measured cell population. In this unit, simple and universally applicable methods for staining fixed cells are presented, as are methods that utilize detergents and/or proteolytic treatment to permeabilize cells and make DNA accessible to fluorochrome...
October 2, 2017: Current Protocols in Cytometry
https://www.readbyqxmd.com/read/28967990/click-chemistry-for-analysis-of-cell-proliferation-in-flow-cytometry
#7
Scott T Clarke, Veronica Calderon, Jolene A Bradford
The measurement of cellular proliferation is fundamental to the assessment of cellular health, genotoxicity, and the evaluation of drug efficacy. Labeling, detection, and quantification of cells in the synthesis phase of cell cycle progression are not only important for characterizing basic biology, but also in defining cellular responses to drug treatments. Changes in DNA replication during S-phase can provide valuable insights into mechanisms of cell growth, cell cycle kinetics, and cytotoxicity. A common method for detection of cell proliferation is the incorporation of a thymidine analog during DNA synthesis...
October 2, 2017: Current Protocols in Cytometry
https://www.readbyqxmd.com/read/28967989/staining-of-frozen-and-formalin-fixed-paraffin-embedded-tissues-with-metal-labeled-antibodies-for-imaging-mass-cytometry-analysis
#8
Qing Chang, Olga Ornatsky, David Hedley
This unit describes protocols for labeling tissue sections using combinations of metal-tagged antibodies and an iridium-containing DNA intercalator for analysis by imaging mass cytometry. Imaging mass cytometry (IMC) allows the labeling of up to 40 individual markers simultaneously using antibody cocktails. We discuss labeling of both cryostat sections and sections from formalin-fixed, paraffin-embedded (FFPE) tissue blocks. The protocols are similar to those used for optical microscopy techniques, while allowing much higher complexity of analysis...
October 2, 2017: Current Protocols in Cytometry
https://www.readbyqxmd.com/read/28967988/cytof-measurement-of-immunocompetence-across-major-immune-cell-types
#9
Priyanka B Subrahmanyam, Holden T Maecker
The central role of the immune system is becoming appreciated in a wide variety of diseases. Cancer immunotherapy is one area that has yielded much recent success, although not all patients benefit equally. At the same time, recent studies have highlighted the heterogeneity of the human immune system. Despite this heterogeneity, we do not routinely measure immune competence in clinical practice, and there are no consensus assays of healthy immune function. Using mass cytometry (CyTOF), we can simultaneously detect ∼40 markers to identify various cell subsets and determine their function by the expression of cytokines, cytotoxicity, and activation markers...
October 2, 2017: Current Protocols in Cytometry
https://www.readbyqxmd.com/read/28678421/cytof-mass-cytometry-for-click-proliferation-assays
#10
Vinko Tosevski, Egor Ulashchik, Andrea Trovato, Paolo Cappella
Novel cell analyzers, including polychromatic flow cytometers and isotopical cytometry by time of flight (CyTOF) mass cytometers, enable simultaneous measurement of virtually bondless characteristics at the single-cell level. BrdU assays for quantifying cellular proliferation are common but have several limitations, including the need for a DNA denaturation step and inability to simultaneously resolve multiple parameters and phenotypic complexity. Click chemistry reactions have become popular in the past decade, as they can resolve these issues...
July 5, 2017: Current Protocols in Cytometry
https://www.readbyqxmd.com/read/28678420/measurement-of-drug-stabilized-topoisomerase-ii-cleavage-complexes-by-flow-cytometry
#11
Marcelo de Campos Nebel, Micaela Palmitelli, Marcela González-Cid
The poisoning of Topoisomerase II (Top2) has been found to be useful as a therapeutic strategy for the treatment of several tumors. The mechanism of Top2 poisons involves a drug-mediated stabilization of a Top2-DNA complex, termed Top2 cleavage complex (Top2cc), which maintains a 5' end of DNA covalently bound to a tyrosine from Top2 through a phosphodiester group. Drug-stabilized Top2cc leads to Top2-linked-DNA breaks, which are believed to mediate their cytotoxicity. Several time-consuming or cell type-limiting assays have been used in the past to study drug-stabilized Top2cc...
July 5, 2017: Current Protocols in Cytometry
https://www.readbyqxmd.com/read/28678419/detection-and-quantification-of-mitochondrial-fusion-using-imaging-flow-cytometry
#12
Aldo Nascimento, Joanne Lannigan, David Kashatus
Mitochondria are dynamic organelles that perform several vital cellular functions. Requisite for these functions are mitochondrial fusion and fission. Despite the increasing importance of mitochondrial dynamics in a range of cellular processes, there exist limited methods for robust quantification of mitochondrial fission and fusion. Currently, the most widely used method to measure mitochondrial fusion is the polyethylene glycol (PEG) fusion assay. While this assay can provide useful information regarding fusion activity, the reliance on manual selection of rare fusion events is time consuming and may introduce selection bias...
July 5, 2017: Current Protocols in Cytometry
https://www.readbyqxmd.com/read/28678418/detection-of-extracellular-vesicles-using-proximity-ligation-assay-with-flow-cytometry-readout-exopla
#13
Liza Löf, Linda Arngården, Tonge Ebai, Ulf Landegren, Ola Söderberg, Masood Kamali-Moghaddam
Extracellular vesicles (EVs) are continuously released by most cells, and they carry surface markers of their cells of origin. Found in all body fluids, EVs function as conveyers of cellular information, and evidence implicates them as markers of disease. These characteristics make EVs attractive diagnostic targets. However, detection and characterization of EVs is challenging due to their small size. We've established a method, called ExoPLA, that allows individual EVs to be detected and characterized at high specificity and sensitivity...
July 5, 2017: Current Protocols in Cytometry
https://www.readbyqxmd.com/read/28678417/stochastic-optical-reconstruction-microscopy-storm
#14
Jianquan Xu, Hongqiang Ma, Yang Liu
Super-resolution (SR) fluorescence microscopy, a class of optical microscopy techniques at a spatial resolution below the diffraction limit, has revolutionized the way we study biology, as recognized by the Nobel Prize in Chemistry in 2014. Stochastic optical reconstruction microscopy (STORM), a widely used SR technique, is based on the principle of single molecule localization. STORM routinely achieves a spatial resolution of 20 to 30 nm, a ten-fold improvement compared to conventional optical microscopy...
July 5, 2017: Current Protocols in Cytometry
https://www.readbyqxmd.com/read/28369766/method-for-dna-ploidy-analysis-along-with-immunophenotyping-for-rare-populations-in-a-sample-using-fxcycle-violet
#15
Prashant Tembhare, Yajamanam Badrinath, Sitaram Ghogale, Papagudi Ganesan Subramanian
The clinical use of flow cytometric DNA ploidy assay has been extended towards stratifying the risk of diseases, such as monoclonal gammopathies or B cell acute lymphoblastic leukemia, and to detect circulating tumor cells, both of which require detection of minute cell populations. This unit describes a protocol for determining DNA ploidy in fixed samples with simultaneous surface immunophenotyping. It is an easy method for simultaneous 6- to 8-color immunophenotyping and DNA content analysis using FxCycle Violet (FCV; DAPI) dye...
April 3, 2017: Current Protocols in Cytometry
https://www.readbyqxmd.com/read/28369765/method-to-detect-the-cellular-source-of-over-activated-nadph-oxidases-using-nad-p-h-fluorescence-lifetime-imaging
#16
Daniel Bremer, Ruth Leben, Ronja Mothes, Helena Radbruch, Raluca Niesner
Fluorescence-lifetime imaging microscopy (FLIM) is a technique to generate images, in which the contrast is obtained by the excited-state lifetime of fluorescent molecules instead of their intensity and emission spectrum. The ubiquitous coenzymes NADH and NADPH, hereafter NAD(P)H, in cells show a short fluorescence lifetime ≈400 psec in the free-state and a longer fluorescence lifetime when bound to enzymes. The fluorescence lifetime of NAD(P)H in this state depends on the binding-site on the specific enzyme...
April 3, 2017: Current Protocols in Cytometry
https://www.readbyqxmd.com/read/28369764/fluorescent-proteins-for-flow-cytometry
#17
Teresa S Hawley, Robert G Hawley, William G Telford
Fluorescent proteins have become standard tools for cell and molecular biologists. The color palette of fluorescent proteins spans the ultraviolet, visible, and near-infrared spectrum. Utility of fluorescent proteins has been greatly facilitated by the availability of compact and affordable solid state lasers capable of providing various excitation wavelengths. In theory, the plethora of fluorescent proteins and lasers make it easy to detect multiple fluorescent proteins simultaneously. However, in practice, heavy spectral overlap due to broad excitation and emission spectra presents a challenge...
April 3, 2017: Current Protocols in Cytometry
https://www.readbyqxmd.com/read/28369763/correlative-fluorescence-and-electron-microscopy-in-3d-scanning-electron-microscope-perspective
#18
Jonathan Franks, Callen T Wallace, Masateru Shibata, Mitsuo Suga, Natasha Erdman, Donna B Stolz, Simon C Watkins
The ability to correlate fluorescence microscopy (FM) and electron microscopy (EM) data obtained on biological (cell and tissue) specimens is essential to bridge the resolution gap between the data obtained by these different imaging techniques. In the past such correlations were limited to either EM navigation in two dimensions to the locations previously highlighted by fluorescence markers, or subsequent high-resolution acquisition of tomographic information using a TEM. We present a novel approach whereby a sample previously investigated by FM is embedded and subjected to sequential mechanical polishing and backscatter imaging by scanning electron microscope...
April 3, 2017: Current Protocols in Cytometry
https://www.readbyqxmd.com/read/28369762/high-throughput-particle-uptake-analysis-by-imaging-flow-cytometry
#19
Asya Smirnov, Michael D Solga, Joanne Lannigan, Alison K Criss
Quantifying the efficiency of particle uptake by host cells is important in the fields of infectious diseases, autoimmunity, cancer, developmental biology, and drug delivery. Here we present a protocol for high-throughput analysis of particle uptake by imaging flow cytometry, using the bacterium Neisseria gonorrhoeae attached to and internalized by neutrophils as an example. Cells are exposed to fluorescently labeled bacteria, fixed, and stained with a bacteria-specific antibody of a different fluorophore. Thus, in the absence of a permeabilizing agent, extracellular bacteria are double-labeled with two fluorophores while intracellular bacteria remain single-labeled...
April 3, 2017: Current Protocols in Cytometry
https://www.readbyqxmd.com/read/28055116/standardization-calibration-and-control-in-flow-cytometry
#20
REVIEW
Lili Wang, Robert A Hoffman
Because flow cytometers are designed to measure particle characteristics, particles are the most common materials used to calibrate, control, and standardize the instruments. Definitions and cautions are provided for common terms to alert the reader to critical distinctions in meaning. This unit presents extensive background on particle types and cautions and describes practical aspects of methods to standardize and calibrate instruments. Procedures are provided to characterize performance in terms of optical alignment, fluorescence and light scatter resolution, and sensitivity...
January 5, 2017: Current Protocols in Cytometry
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