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Current Protocols in Cytometry

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https://www.readbyqxmd.com/read/28967991/analysis-of-cellular-dna-content-by-flow-cytometry
#1
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
#2
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
#3
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
#4
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
#5
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
#6
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
#7
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
#8
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
#9
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
#10
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
#11
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
#12
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
#13
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
#14
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
#15
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
https://www.readbyqxmd.com/read/28055115/measurement-of-t-cell-telomere-length-using-amplified-signal-fish-staining-and-flow-cytometry
#16
Andrea L Henning, Danielle E Levitt, Jakob L Vingren, Brian K McFarlin
Exposure to pathogen-associated molecular patterns (PAMPS), damage-associated molecular patterns (DAMPS), and physiologically challenging stimuli either positively or negatively affect leukocyte maturity. Cellular maturity has implications for the effectiveness of host response to bacterial or viral infection and/or tissue injury. Thus, the ability to accurately assess cellular maturity and health is important to fully understand immune status and function. The most common technique for measuring cellular maturity is to measure telomere length; however, existing techniques are not optimized for single-cell measurements using flow cytometry...
January 5, 2017: Current Protocols in Cytometry
https://www.readbyqxmd.com/read/28055114/quantitative-analysis-of-cellular-senescence-in-culture-and-in-vivo
#17
Jing Zhao, Heike Fuhrmann-Stroissnigg, Aditi U Gurkar, Rafael R Flores, Akaitz Dorronsoro, Donna B Stolz, Claudette M St Croix, Laura J Niedernhofer, Paul D Robbins
Cellular senescence refers to the irreversible growth arrest of normally dividing cells in response to various types of stress. Cellular senescence is induced by telomere shortening due to repeated cell division, which causes a DNA damage response, as well as genotoxic, oxidative, and inflammatory stress. Strong mitogenic signaling, such as oncogene activation, also drives cells into a senescent state. Senescent cells express a specific subset of genes, termed the senescence-associated secretory phenotype (SASP), including pro-inflammatory factors, growth factors, and matrix metalloproteinases, which together promote non-cell autonomous, secondary senescence...
January 5, 2017: Current Protocols in Cytometry
https://www.readbyqxmd.com/read/28055113/assessment-of-telomere-length-phenotype-and-dna-content
#18
Theodoros Kelesidis, Ingrid Schmid
Telomere sequences at the end of chromosomes control somatic cell division; therefore, telomere length in a given cell population provides information about its replication potential. This unit describes a method for flow cytometric measurement of telomere length in subpopulations using fluorescence in situ hybridization of fluorescently-labeled probes (Flow-FISH) without prior cell separation. After cells are stained for surface immunofluorescence, antigen-antibody complexes are covalently cross-linked onto cell membranes before FISH with a telomere-specific probe...
January 5, 2017: Current Protocols in Cytometry
https://www.readbyqxmd.com/read/28055112/identification-of-human-memory-like-nk-cells
#19
Elena I Kovalenko, Maria A Streltsova, Leonid M Kanevskiy, Sophia A Erokhina, William G Telford
Our understanding of NK biology is increased dramatically, a product of improved flow-cytometric techniques for analyzing these cells. NK cells undergo significant changes in repertoire during differentiation. A repeating stimulus, such as a cytomegalovirus infection, may result in accumulation of certain types of highly differentiated NK cells designated as memory-like, or adaptive NK cells. Adaptive NK cells are capable of rapid expansion and effective response to the recall stimulus. These cells differ significantly from conventional NK cells both functionally and phenotypically...
January 5, 2017: Current Protocols in Cytometry
https://www.readbyqxmd.com/read/27723090/flow-analysis-and-sorting-of-plant-chromosomes
#20
Jan Vrána, Petr Cápal, Hana Šimková, Miroslava Karafiátová, Jana Čížková, Jaroslav Doležel
Analysis and sorting of plant chromosomes (plant flow cytogenetics) is a special application of flow cytometry in plant genomics and its success depends critically on sample quality. This unit describes the methodology in a stepwise manner, starting with the induction of cell cycle synchrony and accumulation of dividing cells in mitotic metaphase, and continues with the preparation of suspensions of intact mitotic chromosomes, flow analysis and sorting of chromosomes, and finally processing of the sorted chromosomes...
October 10, 2016: Current Protocols in Cytometry
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