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Current Protocols in Cytometry

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
Anja J Gerrits, Andrew L Frelinger, Alan D Michelson
In inflammatory and thrombotic syndromes, platelets aggregate with circulating leukocytes, especially monocytes and neutrophils. This leukocyte-platelet aggregate formation is initiated primarily through platelet surface expression of P-selectin (CD62P), following activation-dependent degranulation of α-granules, binding to its constitutively expressed counter-receptor, P-selectin glycoprotein ligand 1 (PSGL-1), on leukocytes. Monocyte-platelet aggregates are a more sensitive marker of platelet activation than platelet surface P-selectin...
October 10, 2016: Current Protocols in Cytometry
Neil J Kelly, Nadine Dandachi, Dmitry A Goncharov, Andressa Z Pena, Josiah E Radder, Alyssa D Gregory, Yen-Chun Lai, Adriana S Leme, Mark T Gladwin, Elena A Goncharova, Claudette M St Croix, Steven D Shapiro
The quantification of tunica media thickness in histological cross sections is a ubiquitous exercise in cardiopulmonary research, yet the methods for quantifying medial wall thickness have never been rigorously examined with modern image analysis tools. As a result, inaccurate and cumbersome manual measurements of discrete wall regions along the vessel periphery have become common practice for wall thickness quantification. The aim of this study is to introduce, validate, and facilitate the use of an improved method for medial wall thickness quantification...
October 10, 2016: Current Protocols in Cytometry
William Telford, Karen Tamul, Jolene Bradford
Apoptosis is an important mechanism in cell biology, playing a critical regulatory role in virtually every organ system. It has been particularly well characterized in the immune system, with roles ranging from immature immune cell development and selection to down-regulation of the mature immune response. Apoptosis is also the primary mechanism of action of anti-cancer drugs. Flow cytometry has been the method of choice for analyzing apoptosis in suspension cells for more than 25 years. Numerous assays have been devised to measure both the earliest and latest steps in the apoptotic process, from the earliest signal-transduction events to the late morphological changes in cell shape and granularity, proteolysis, and chromatin condensation...
July 1, 2016: Current Protocols in Cytometry
Kah Teong Soh, Joseph D Tario, Sean Colligan, Orla Maguire, Dalin Pan, Hans Minderman, Paul K Wallace
Nucleic acid content can be quantified by flow cytometry through the use of intercalating compounds; however, measuring the presence of specific sequences has hitherto been difficult to achieve by this methodology. The primary obstacle to detecting discrete nucleic acid sequences by flow cytometry is their low quantity and the presence of high background signals, rendering the detection of hybridized fluorescent probes challenging. Amplification of nucleic acid sequences by molecular techniques such as in situ PCR have been applied to single-cell suspensions, but these approaches have not been easily adapted to conventional flow cytometry...
January 6, 2016: Current Protocols in Cytometry
Véronique Wuyts, Nancy H C Roosens, Sophie Bertrand, Kathleen Marchal, Sigrid C J De Keersmaecker
Characterization of microbial pathogens is necessary for surveillance, outbreak detection, and tracing of outbreak sources. This unit describes a multiplex oligonucleotide ligation-PCR (MOL-PCR) optimized for characterization of microbial pathogens. With MOL-PCR, different types of markers, like unique sequences, single-nucleotide polymorphisms (SNPs) and indels, can be simultaneously analyzed in one assay. This assay consists of a multiplex ligation for detection of the markers, a singleplex PCR for signal amplification, and hybridization to MagPlex-TAG beads for readout on a Luminex platform after fluorescent staining...
January 6, 2016: Current Protocols in Cytometry
Torsten Kroll, David Schmidt, Georg Schwanitz, Mubashir Ahmad, Jana Hamann, Corinne Schlosser, Yu-Chieh Lin, Konrad J Böhm, Jan Tuckermann, Aspasia Ploubidou
High-content analysis (HCA) converts raw light microscopy images to quantitative data through the automated extraction, multiparametric analysis, and classification of the relevant information content. Combined with automated high-throughput image acquisition, HCA applied to the screening of chemicals or RNAi-reagents is termed high-content screening (HCS). Its power in quantifying cell phenotypes makes HCA applicable also to routine microscopy. However, developing effective HCA and bioinformatic analysis pipelines for acquisition of biologically meaningful data in HCS is challenging...
2016: Current Protocols in Cytometry
Aaron B Kantor, Wayne A Moore, Stephen Meehan, David R Parks
We present a quantitative method for comparing the brightness of antibody-dye reagents and estimating antibodies bound per cell. The method is based on complementary binding of test and fill reagents to antibody capture microspheres. Several aliquots of antibody capture beads are stained with varying amounts of the test conjugate. The remaining binding sites on the beads are then filled with a second conjugate containing a different fluorophore. Finally, the fluorescence of the test conjugate compared to the fill conjugate is used to measure the relative brightness of the test conjugate...
2016: Current Protocols in Cytometry
Andrea L Henning, Jill N Best Sampson, Brian Keith McFarlin
Recent advances in instrument design and reagent development have enabled the rapid progression in available measurement techniques in the field of flow cytometry. In particular, image-based flow cytometry extends the analysis capacity found in traditional flow cytometry. Until recently, it was not possible to measure intracellular mRNA in specific phenotypes of cells by flow cytometry. In this protocol, a method of completing simultaneous intracellular measurement of mRNA and protein for PPAR-gamma in peripheral blood monocytes, which have been exposed in vitro to modified LDL, is described...
2016: Current Protocols in Cytometry
Alfonso Schmidt, Tiffany Bouchery, Graham Le Gros, Kylie M Price
Traditional jet-in-air cell sorters have been designed and optimized to isolate small particles such as mammalian lymphocytes with an average diameter of 10 μm. We discuss the practical considerations of setting up a conventional jet-in-air cell sorter, using a 200-μm nozzle, to isolate the large parasitic nematode eggs of Nippostrongylus brasiliensis, with a maximum size of 60 μm. The eggs were separated based on light scattering properties, no fluorescent dye or molecule was required.
2016: Current Protocols in Cytometry
Lili Wang, Heba Degheidy, Fatima Abbasi, Howard Mostowski, Gerald Marti, Steven Bauer, Robert A Hoffman, Adolfas K Gaigalas
Multicolor flow cytometer assays with fluorescently labeled antibodies are routinely used in clinical laboratories to measure the cell number of specific immunophenotypes and to estimate expression levels of specific receptors/antigens either on the cell surface or intracellularly. The cell number and specific receptors/antigens serve as biomarkers for pathological conditions at various stages of a disease. Existing methods and cell reference materials for quantitative expression measurements have not yet produced results that are of wide clinical interest or are instrument-independent across all fluorescence channels...
2016: Current Protocols in Cytometry
Nicholas K H Khoo, Nadiezhda Cantu-Medellin, Claudette St Croix, Eric E Kelley
A plethora of disease processes are associated with elevated reactive species formation and allied reactions with biomolecules that alter cell signaling, induce overt damage, and promote dysfunction of tissues. Unfortunately, effective detection of reactive species in tissues is wrought with issues that significantly limit capacity for validating species identity, establishing accurate concentrations, and identifying anatomic sites of production. These shortcomings reveal the pressing need for new approaches to more precisely assess reactive species generation in vivo...
October 1, 2015: Current Protocols in Cytometry
Cliff J Luke, Linda P O'Reilly
Caenorhabditis elegans is a powerful model organism for studying human biology and disease due to its surprisingly high genetic homology to Homo sapiens. Its genetic amenability, small size, short generation time, and transparent body make it an ideal organism for multiple scientific disciplines. Fluorescent microscopy is essential for studying protein biological function. However, C. elegans, mainly due to its high motility, has been more difficult to adapt to fluorescence imaging, especially live-imaging...
October 1, 2015: Current Protocols in 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 glucose-supplemented medium and exposed to increasing concentrations of various chemicals. Several key mitochondrial/cellular parameters known to be directly impacted by mitochondrial dysfunction, such as mitochondrial membrane potential (MMP), mitochondrial reactive oxygen species (ROS) production, intracellular reduced glutathione (GSH) level, and cell viability, are simultaneously measured by flow cytometry...
2015: Current Protocols in Cytometry
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 protocols for collection of blood, separation of leukocytes from whole blood by lysis of erythrocytes, isolating mononuclear cells by density gradient separation, and assorted non-flow sorting methods, such as magnetic bead separations, for enriching specific cell populations, including monocytes, T lymphocytes, B lymphocytes, neutrophils, and platelets, prior to flow cytometric analysis...
2015: Current Protocols in Cytometry
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 development of improved approaches to EV detection and characterization will be critical to further understanding their roles in physiology and disease. Flow cytometry has been a popular tool for measuring cell-derived EVs, but has often been used in an uncritical manner in which fundamental principles and limitations of the instrument are ignored...
2015: Current Protocols in Cytometry
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 telomeres within promyelocytic leukemia (PML) nuclear bodies, which harbor proteins that are linked with DNA repair and recombination activity. For this reason, it is speculated that these associated PML bodies represent the sites of the recombination that maintains telomere length...
2015: Current Protocols in 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. Most earlier techniques for measuring cellular activation parameters determined the mean value for a population of cells, which did not permit optimal resolution of the responses. The flow cytometer is particularly useful for this purpose because it can measure ion concentrations in large numbers of single cells and thereby allows ion concentration to be correlated with other parameters such as immunophenotype and cell cycle stage...
2015: Current Protocols in Cytometry
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. However, the protocols are often challenging to follow, partly due to difficulties related to sample preparation, instrument parameters, data display, and selection strategies. In addition, troubleshooting of flow cytometry experiments usually requires some fluency in technical principles and instrument specifications and settings. This unit describes setup and procedures for analysis and sorting of male meiotic prophase I (MPI) cells and other germ cells...
2015: Current Protocols in Cytometry
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. However, DNA denaturation is required for BrdU detection with the consequence that most protein epitopes are destroyed and their immunocytochemical detection for multiplex analysis is not possible. A novel assay is presented for identifying cells in active S-phase that does not require the DNA denaturation step but nevertheless detects BrdU. For this purpose, cells were pulsed for a short time by 5-ethynyl-2'-deoxyuridine (EdU) which is incorporated into DNA...
2015: Current Protocols in Cytometry
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