Titration of fluorochrome-conjugated antibodies for labeling cell surface markers on live cells.
Authors: Hulspas R
Abstract
Nonspecific antibody binding is best eliminated by optimizing the amount and concentration of the antibody. An antibody titration assay should be applied to determine the antibody amount and concentration resulting in the highest signal of the positive population and the lowest signal of the negative population. While conventional antibody titration protocols focus on the concentration of the antibody, this protocol for antibody titration considers the antibody concentration, as well as the antibody amount. Thus, it is designed to find the optimal antibody concentration for labe...
Source: Current Protocols in Cytometry - November 20, 2015 Category: Molecular Biology Tags: Curr Protoc Cytom Source Type: research
Phenotypic analysis using very small volumes of blood.
Authors: Weaver JL, McKinnon K, Germolec DR
Abstract
Analysis of cell-surface phenotype of peripheral blood leukocytes is one of the most common applications of flow cytometry. In mouse research, the small size of the animal limits the amount of blood available. Standard staining methods using lysis of erythrocytes or gradient separation followed by repeated washing involve unavoidable losses of cells that generally limit analysis of blood to terminal methods. Time-course studies, therefore, require sacrifice of groups of mice at each time point. Thus, a method is needed that can be used with much smaller ...
Source: Current Protocols in Cytometry - November 20, 2015 Category: Molecular Biology Tags: Curr Protoc Cytom Source Type: research
Assessment of beta cell viability.
Authors: Jayaraman S
Abstract
This unit contains detailed protocols for the simultaneous identification of the human pancreatic β cells and determination of their viability by flow cytometry. The enumeration of β cells is based on the ability of the cell-permeable form of the zinc-selective dye, FluoZin-3-AM, to bind intracellular labile zinc stored at higher levels in these cells than any other types of cells in the body. Although staining of intracellular labile zinc by FluoZin-3-AM is dependent on the metabolic activity of β cells, co-staining with a mitochondrial transmembrane potential indicator al...
Source: Current Protocols in Cytometry - November 20, 2015 Category: Molecular Biology Tags: Curr Protoc Cytom Source Type: research
Fluorescent cell barcoding for multiplex flow cytometry.
Authors: Krutzik PO, Clutter MR, Trejo A, Nolan GP
Abstract
Fluorescent cell barcoding (FCB) enables high throughput, high content flow cytometry by multiplexing samples prior to staining and acquisition on the cytometer. Individual cell samples are barcoded, or labeled, with unique signatures of fluorescent dyes so that they can be mixed together, stained, and analyzed as a single sample. By mixing samples prior to staining, antibody consumption is typically reduced 10- to 100-fold. In addition, data robustness is increased through the combination of control and treated samples, which minimizes pipetting ...
Source: Current Protocols in Cytometry - November 20, 2015 Category: Molecular Biology Tags: Curr Protoc Cytom Source Type: research
High-resolution multiparameter DNA flow cytometry for the detection and sorting of tumor and stromal subpopulations from paraffin-embedded tissues.
Authors: Corver WE, ter Haar NT
Abstract
This unit contains a detailed protocol for the simultaneous flow cytometric measurement of tumor cells, stromal cells, and DNA content of formalin-fixed, paraffin-embedded (FFPE) tissues. The vimentin-positive stromal cell fraction can be used as an internal reference for DNA content assessments. This allows clear detection of keratin-positive tumor cells with a DNA index lower than 1.0 and of keratin-positive tumor cells with a DNA close to 1.0 in overall DNA aneuploid samples, thus improved DNA ploidy assessment in FFPE carcinomas. Furthermore, the protocol is use...
Source: Current Protocols in Cytometry - November 20, 2015 Category: Molecular Biology Tags: Curr Protoc Cytom Source Type: research
Dual-pulse labeling using 5-ethynyl-2'-deoxyuridine (EdU) and 5-bromo-2'-deoxyuridine (BrdU) in flow cytometry.
Authors: Bradford JA, Clarke ST
Abstract
Changes in DNA replication during S-phase can give insights into mechanisms of cell growth, cell cycle kinetics, and cytotoxicity. A common method for detection of cell proliferation utilizes the incorporation of a thymidine analog during DNA synthesis. Incorporation of multiple analogs at different time points can further define cell cycle kinetics. Traditionally, the dual-pulse method has been done by combining 5-bromo-2'-deoxyuridine (BrdU) with iododeoxyuridine or chlorodeoxyuridine, with detection using multiple cross-reacting BrdU antibodies. This unit present...
Source: Current Protocols in Cytometry - November 20, 2015 Category: Molecular Biology Tags: Curr Protoc Cytom Source Type: research
Optical filters for wavelength selection in fluorescence instrumentation.
Authors: Erdogan T
Abstract
Fluorescence imaging and analysis techniques have become ubiquitous in life science research, and they are poised to play an equally vital role in in vitro diagnostics (IVD) in the future. Optical filters are crucial for nearly all fluorescence microscopes and instruments, not only to provide the obvious function of spectral control, but also to ensure the highest possible detection sensitivity and imaging resolution. Filters make it possible for the sample to "see" light within only the absorption band, and the detector to "see" light within only the emission band. Without filt...
Source: Current Protocols in Cytometry - November 20, 2015 Category: Molecular Biology Tags: Curr Protoc Cytom Source Type: research
Quantitative assessment of pancreatic islets using laser scanning cytometry.
Authors: Krull D
Abstract
Insulin-dependent (type II) diabetes is characterized by an inability to metabolize glucose, resulting from insufficient insulin function for glucose transport from the blood to tissues. One cause of insufficiency is malfunction of the insulin-producing beta cells within the pancreatic islets. Various compounds to stimulate and restore normal islet function are under development. Zucker diabetic fatty (ZDF) rat animal models are used to measure efficacy of drug candidates, as they show clinical effects similar to those in diabetic patients. Drug effects are evaluated by removing t...
Source: Current Protocols in Cytometry - November 20, 2015 Category: Molecular Biology Tags: Curr Protoc Cytom Source Type: research
Critical aspects in analysis of cellular DNA content.
Authors: Darzynkiewicz Z
Abstract
This unit covers general aspects of DNA content analysis and provides introductory or complementary information to the specific protocols of DNA content assessment in this chapter. It describes principles of DNA content analysis and outlines difficulties and pitfalls common to these methods. It also reviews methods of DNA staining in live, permeabilized, and fixed cells, and in cell nuclei isolated from paraffin-embedded tissues, as well as the approaches to stain DNA concurrently with cell immunophenotype. This unit addresses factors affecting accuracy of DNA measurement,...
Source: Current Protocols in Cytometry - November 20, 2015 Category: Molecular Biology Tags: Curr Protoc Cytom Source Type: research
High-resolution cell cycle and DNA ploidy analysis in tissue samples.
Authors: Heinlein C, Speidel D
Abstract
This unit describes an easy, rapid, and universal procedure to process fresh and nitrogen-frozen tissue specimens for high-resolution cell cycle and DNA ploidy analysis. Unlike other protocols, this procedure does not require treating tissues with enzymes, detergents, or other plasma membrane-lysing chemicals, but it achieves tissue dispersion by a simple two-step mechanical process that can be performed in ∼5 min. Resulting single-cell suspensions are fixed with ethanol, stained with propidium iodide, and subjected to flow cytometric DNA content analysis. The meth...
Source: Current Protocols in Cytometry - November 20, 2015 Category: Molecular Biology Tags: Curr Protoc Cytom Source Type: research
In situ proximity ligation assay for microscopy and flow cytometry.
Authors: Leuchowius KJ, Weibrecht I, Söderberg O
Abstract
The ability to study proteins and protein interactions inside cells and tissues is important for elucidating how cells function in health and disease. The in situ proximity ligation assay (in situ PLA) presented here can be used to visualize proteins, protein-protein interactions, and post-translational modifications in cells and tissues. The method is based upon the use of antibodies that target the proteins involved in an interaction; hence, the method has the advantage that it can be used in clinical specimens, providing localized, quantifiable ...
Source: Current Protocols in Cytometry - November 20, 2015 Category: Molecular Biology Tags: Curr Protoc Cytom Source Type: research
Setting up and running an advanced light microscopy and imaging facility.
Authors: Sánchez C, Muñoz MÁ, Villalba M, Labrador V, Díez-Guerra FJ
Abstract
During the last twenty years, interest in light microscopy and imaging techniques has grown in various fields, such as molecular and cellular biology, developmental biology, and neurobiology. In addition, the number of scientific articles and journals using these techniques is rapidly increasing. Nowadays, most research institutions require sophisticated microscopy systems to cover their investigation demands. In general, such instruments are too expensive and complex to be purchased and managed by a single laboratory or rese...
Source: Current Protocols in Cytometry - November 20, 2015 Category: Molecular Biology Tags: Curr Protoc Cytom Source Type: research
Photoactivation and imaging of optical highlighter fluorescent proteins.
Authors: Patterson GH
Abstract
A major advance in the microscopic study of cells and tissues is the introduction of photoactivatable fluorescent proteins, which can specifically mark proteins of interest within a living cell. Fluorescent proteins are now available that allow a pool of molecules to be "turned on" by photoactivation. This unit discusses technical aspects for the general use of photoactivatable fluorescent proteins and introduces some specific applications in the concluding remarks.
PMID: 21732309 [PubMed - indexed for MEDLINE] (Source: Current Protocols in Cytometry)
Source: Current Protocols in Cytometry - November 20, 2015 Category: Molecular Biology Tags: Curr Protoc Cytom Source Type: research
Zinc fixation for flow cytometry analysis of intracellular and surface epitopes, DNA content, and cell proliferation.
Authors: Christensen R, Owens DM, Thomsen A, Pedersen S, Jensen UB
Abstract
Zinc salt-based fixation (ZBF) is a simple, cost-effective, and nonhazardous fixation method for cell suspensions that preserves all cellular structures and enables flow cytometric analysis of both surface and intracellular proteins, DNA content profiles, and pulse-labeling using the thymidine analog EdU in the same cell sample. ZBF performs equally well to formaldehyde in the preservation of surface epitope labeling and forward and side light scatter parameters, as measured by flow cytometry. DNA is maintained at high molecular we...
Source: Current Protocols in Cytometry - November 20, 2015 Category: Molecular Biology Tags: Curr Protoc Cytom Source Type: research
Microscope objectives.
The objective is the most crucial image-forming component of a microscope. A knowledge of the many types of objectives available and their characteristics is critical to the selection of appropriate objectives for image cytometry. This unit discusses aberrations in image formation and their correction, construction, and types of objectives, and objectives for other microscopy applications, explaining the advantages and limitations of each one.
PMID: 21965106 [PubMed - indexed for MEDLINE] (Source: Current Protocols in Cytometry)
Source: Current Protocols in Cytometry - November 20, 2015 Category: Molecular Biology Tags: Curr Protoc Cytom Source Type: research
