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Current Protocols in Cytometry

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
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
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
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
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
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
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...
April 1, 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.
April 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
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...
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
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
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...
July 1, 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...
July 1, 2015: Current Protocols in Cytometry
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