Click Chemistry for Analysis of Cell Proliferation in Flow Cytometry.
Authors: Clarke ST, Calderon V, Bradford JA
Abstract
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 t...
Source: Current Protocols in Cytometry - October 4, 2017 Category: Molecular Biology Tags: Curr Protoc Cytom Source Type: research
Analysis of Cellular DNA Content by Flow Cytometry.
Authors: Darzynkiewicz Z, Huang X, Zhao H
Abstract
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. Additionally, supravital cell staining with Hoe...
Source: Current Protocols in Cytometry - October 4, 2017 Category: Molecular Biology Tags: Curr Protoc Cytom Source Type: research
Stochastic Optical Reconstruction Microscopy (STORM).
Authors: Xu J, Ma H, Liu Y
Abstract
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. Among all SR techniques, STORM offers a high spatial resolution ...
Source: Current Protocols in Cytometry - July 6, 2017 Category: Molecular Biology Tags: Curr Protoc Cytom Source Type: research
Detection of Extracellular Vesicles Using Proximity Ligation Assay with Flow Cytometry Readout-ExoPLA.
We describe the procedures for ExoPLA, along with expected results and troubleshooting. © 2017 by John Wiley & Sons, Inc.
PMID: 28678418 [PubMed - in process] (Source: Current Protocols in Cytometry)
Source: Current Protocols in Cytometry - July 6, 2017 Category: Molecular Biology Tags: Curr Protoc Cytom Source Type: research
Detection and Quantification of Mitochondrial Fusion Using Imaging Flow Cytometry.
Authors: Nascimento A, Lannigan J, Kashatus D
Abstract
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 select...
Source: Current Protocols in Cytometry - July 6, 2017 Category: Molecular Biology Tags: Curr Protoc Cytom Source Type: research
Measurement of Drug-Stabilized Topoisomerase II Cleavage Complexes by Flow Cytometry.
Authors: de Campos Nebel M, Palmitelli M, González-Cid M
Abstract
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 th...
Source: Current Protocols in Cytometry - July 6, 2017 Category: Molecular Biology Tags: Curr Protoc Cytom Source Type: research
CyTOF Mass Cytometry for Click Proliferation Assays.
Authors: Tosevski V, Ulashchik E, Trovato A, Cappella P
Abstract
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 issu...
Source: Current Protocols in Cytometry - July 6, 2017 Category: Molecular Biology Tags: Curr Protoc Cytom Source Type: research
High-Throughput Particle Uptake Analysis by Imaging Flow Cytometry.
Authors: Smirnov A, Solga MD, Lannigan J, Criss AK
Abstract
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 ...
Source: Current Protocols in Cytometry - April 4, 2017 Category: Molecular Biology Tags: Curr Protoc Cytom Source Type: research
Correlative Fluorescence and Electron Microscopy in 3D-Scanning Electron Microscope Perspective.
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. The resulting three dimensional EM tomogram of the sample can be directly correlated to the FM data. © 2017 by John Wiley & Sons, Inc.
PMID: 28369763 [PubMed - in process] (Source: Current Protocols in Cytometry)
Source: Current Protocols in Cytometry - April 4, 2017 Category: Molecular Biology Tags: Curr Protoc Cytom Source Type: research
Fluorescent Proteins for Flow Cytometry.
Authors: Hawley TS, Hawley RG, Telford WG
Abstract
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 a...
Source: Current Protocols in Cytometry - April 4, 2017 Category: Molecular Biology Tags: Curr Protoc Cytom Source Type: research
Method to Detect the Cellular Source of Over-Activated NADPH Oxidases Using NAD(P)H Fluorescence Lifetime Imaging.
Authors: Bremer D, Leben R, Mothes R, Radbruch H, Niesner R
Abstract
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. In the case of NADPH bound ...
Source: Current Protocols in Cytometry - April 4, 2017 Category: Molecular Biology Tags: Curr Protoc Cytom Source Type: research
Method for DNA Ploidy Analysis Along with Immunophenotyping for Rare Populations in a Sample using FxCycle Violet.
Authors: Tembhare P, Badrinath Y, Ghogale S, Subramanian PG
Abstract
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. This pr...
Source: Current Protocols in Cytometry - April 4, 2017 Category: Molecular Biology Tags: Curr Protoc Cytom Source Type: research
Identification of Human Memory-Like NK Cells.
Authors: Kovalenko EI, Streltsova MA, Kanevskiy LM, Erokhina SA, Telford WG
Abstract
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 fro...
Source: Current Protocols in Cytometry - January 6, 2017 Category: Molecular Biology Tags: Curr Protoc Cytom Source Type: research
Assessment of Telomere Length, Phenotype, and DNA Content.
Authors: Kelesidis T, Schmid I
Abstract
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. ...
Source: Current Protocols in Cytometry - January 6, 2017 Category: Molecular Biology Tags: Curr Protoc Cytom Source Type: research
Quantitative Analysis of Cellular Senescence in Culture and In Vivo.
Authors: Zhao J, Fuhrmann-Stroissnigg H, Gurkar AU, Flores RR, Dorronsoro A, Stolz DB, St Croix CM, Niedernhofer LJ, Robbins PD
Abstract
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 senesce...
Source: Current Protocols in Cytometry - January 6, 2017 Category: Molecular Biology Tags: Curr Protoc Cytom Source Type: research
