Flow cytometry of murine spermatocytes.
Authors: Gaysinskaya V, Bortvin A
Abstract
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...
Source: Current Protocols in Cytometry - November 20, 2015 Category: Molecular Biology Tags: Curr Protoc Cytom Source Type: research
Measurement of intracellular ions by flow cytometry.
Authors: Posey AD, Kawalekar OU, June CH
Abstract
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 a...
Source: Current Protocols in Cytometry - November 20, 2015 Category: Molecular Biology Tags: Curr Protoc Cytom Source Type: research
Visualization of Telomere Integrity and Function In Vitro and In Vivo Using Immunofluorescence Techniques.
Authors: Cesare AJ, Heaphy CM, O'Sullivan RJ
Abstract
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 maint...
Source: Current Protocols in Cytometry - November 20, 2015 Category: Molecular Biology Tags: Curr Protoc Cytom Source Type: research
Flow Cytometry of Extracellular Vesicles: Potential, Pitfalls, and Prospects.
Authors: Nolan JP
Abstract
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 fundamen...
Source: Current Protocols in Cytometry - November 20, 2015 Category: Molecular Biology Tags: Curr Protoc Cytom Source Type: research
Collection, Storage, and Preparation of Human Blood Cells.
Authors: Dagur PK, McCoy JP
Abstract
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,...
Source: Current Protocols in Cytometry - November 20, 2015 Category: Molecular Biology Tags: Curr Protoc Cytom Source Type: research
High Throughput-Based Mitochondrial Function Assays by Multi-Parametric Flow Cytometry.
Authors: Robinson JP, Li N, Narayanan PK
Abstract
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 react...
Source: Current Protocols in Cytometry - November 20, 2015 Category: Molecular Biology Tags: Curr Protoc Cytom Source Type: research
Microscopic Investigation of Protein Function in C. elegans Using Fluorescent Imaging.
We present here several protocols for the study of protein location, function and dynamics in context of a whole animal. These protocols, especially when combined with existing genetic procedures, can yield a great deal of insight in the physiological roles of proteins in C. elegans, which can be directly translated into mammalian systems. © 2015 by John Wiley & Sons, Inc.
PMID: 26423692 [PubMed - in process] (Source: Current Protocols in Cytometry)
Source: Current Protocols in Cytometry - November 20, 2015 Category: Molecular Biology Tags: Curr Protoc Cytom Source Type: research
In Vivo Immuno-Spin Trapping: Imaging the Footprints of Oxidative Stress.
Authors: Khoo NK, Cantu-Medellin N, St Croix C, Kelley EE
Abstract
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 reactiv...
Source: Current Protocols in Cytometry - November 20, 2015 Category: Molecular Biology Tags: Curr Protoc Cytom Source Type: research
