Current Protocols in Cytometry最新文献

Flow cytometry is the method of choice to ‘diagnose’ paroxysmal nocturnal hemoglobinuria (PNH) and has led to improved patient management. Most laboratories have limited experience with PNH testing, and many different flow approaches are used. Careful selection and validation of antibody conjugates has allowed the development of reagent cocktails suitable for detection of PNH RBCs, CD71+ reticulocytes, and WBCs in clinical/sub-clinical PNH samples. A CD235a-FITC/CD59-PE assay was developed capable of detecting Type III PNH RBCs at 0.01% sensitivity. A protocol targeting immature CD71+ RBCs can detect PNH reticulocytes at similar sensitivity. Four-color FLAER-based neutrophil and monocyte assays were developed to detect PNH phenotypes at a level of 0.01% and 0.04% sensitivity, respectively. For instrumentation with five or more PMTs, a single-tube 5-color FLAER/CD157-based assay to simultaneously detect PNH neutrophils and monocytes is described. Using these standardized approaches, results have demonstrated good intra- and inter-laboratory performance characteristics even in laboratories with little prior experience performing PNH testing. © 2015 by John Wiley & Sons, Inc.

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. Included are procedures that guide data acquisition, display, gating, and back-gating critical for optimal data visualization and cell sorting. Additionally, a flow cytometry analysis of spermatogenesis-defective testis is provided to illustrate the applicability of the technique to the characterization and purification of cells from mutant testis. © 2015 by John Wiley & Sons, Inc.

The lipophilic cation JC-1 (5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl-benzimidazolyl carbocyanine iodide) has been used for more than 20 years as a specific dye for measuring mitochondrial membrane potential (ΔΨ_m). In this unit, we revise our original protocol (that made use of a single 488 nm laser for the detection of monomers and aggregates, and where compensation was an important step) to use dual-laser excitation. Moreover, thanks to recently developed multilaser instruments and novel probes for surface and intracellular markers, JC-1 can be utilized by polychromatic flow cytometry to simultaneously detect, without any compensation between fluorescences, ΔΨ_m along with other biological parameters, such as apoptosis and the production of reactive oxygen species. © 2015 by John Wiley & Sons, Inc.

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. The nucleotide-exposed ethynyl residue was then derivatized by a copper-catalyzed cycloaddition reaction (“click chemistry” coupling) using a BrdU azide probe. The resulting DNA-bound bromouracil moieties were then detected by commercial anti-BrdU monoclonal antibodies without the need for a denaturation step. This method has been tested using several cell lines and is more sensitive than traditional BrdU and allows multicolor and multiplex analysis in flow cytometry (FCM) and image-based cytometry. © 2015 by John Wiley & Sons, Inc.

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. A limitation of flow cytometry, however, is that it does not permit resolution of certain complex kinetic responses such as cellular oscillatory responses. This unit describes the preparation of cells, including labeling with antibodies and with calcium probes, and discusses the principles of data analysis and interpretation. © 2015 by John Wiley & Sons, Inc.

Cell volume is an important parameter in cell adaptation to anisosmotic stress, in the development of apoptosis and necrosis, and in the pathogenesis of several diseases. This unit describes a method for measuring the volume of adherent cells using a standard light microscope. A coverslip with attached cells is placed in a shallow chamber in a medium containing a strongly absorbing and cell-impermeant dye, Acid Blue 9. When such a sample is imaged in transmitted light at a wavelength of maximum dye absorption (630 nm), the resulting contrast quantitatively reflects cell thickness. Once the thickness is known at every point, the volume can be computed as well. Technical details, interpretation of data, and possible artifacts are discussed. Measurements in absolute units require knowledge of the absorption coefficient, and a similar procedure for the measurement of absorption coefficient is described. © 2015 by John Wiley & Sons, Inc.

The development of confocal microscopy techniques introduced the ability to optically section fluorescent samples in the axial dimension, perpendicular to the image plane. These approaches, via the placement of a pinhole in the conjugate image plane, provided superior resolution in the axial (z) dimension resulting in nearly isotropic optical sections. However, increased axial resolution, via pinhole optics, comes at the cost of both speed and excitation efficiency. Light sheet fluorescent microscopy (LSFM), a century-old idea made possible with modern developments in both excitation and detection optics, provides sub-cellular resolution and optical sectioning capabilities without compromising speed or excitation efficiency. Over the past decade, several variations of LSFM have been implemented each with its own benefits and deficiencies. Here we discuss LSFM fundamentals and outline the basic principles of several major light-sheet-based imaging modalities (SPIM, inverted SPIM, multi-view SPIM, Bessel beam SPIM, and stimulated emission depletion SPIM) while considering their biological relevance in terms of intrusiveness, temporal resolution, and sample requirements. © 2015 by John Wiley & Sons, Inc.

Mixtures of ascorbate and copper used in certain click chemistry experimental conditions act as oxidizing agents, catalyzing the formation of reactive oxygen species through Fenton and related reactions. Hydroxyl radicals act as chemical nucleases, introducing DNA strand breaks that can be exploited for BrdU immunostaining in place of acid denaturation. This procedure is readily applicable to high content analysis and flow cytometry assays, and provides results comparable to click chemistry EdU cycloaddition and classical BrdU immunodetection. Importantly, this approach allows preservation of labile epitopes such as phosphoproteins. This unit describes an optimized method that successfully employs Fenton chemistry for simultaneous detection of phosphoproteins and BrdU in intact cells. © 2015 by John Wiley & Sons, Inc.

Cytokine-producing cells are at the center of the adaptive immune responses, and quantifying these cells is an important aspect to build understanding of the immune response. In particular, Th1 and Th17 cells have been implicated in the pathogenesis of such diseases as inflammatory bowel disease, rheumatoid arthritis, and multiple sclerosis. Quantification of Th1 and Th17 cells can provide important information in research of these diseases and other Th1- and Th17-mediated immune disorders. In vitro stimulation of cells followed by surface and intracellular staining, presented here, has the advantage of detecting the cytokines directly instead of relying exclusively on surrogate surface markers which, although showing enrichment for the effector T cells, are not specific markers for the cytokine-producing cells. © 2015 by John Wiley & Sons, Inc.

Rapid progress is being made to understand the regulatory mechanisms that underlie the epigenetic control of gene expression through histone modification. It is now recognized that this plays a major role in normal development and disease. This unit describes the application of flow cytometry to the study of epigenetic mechanisms by combining labeling of individual histone modifications and phenotypic markers, and it also discusses practical issues to optimize staining. The focus is on normal blood and samples from leukemia patients, but it can also be applied to cells grown in tissue culture. © 2015 by John Wiley & Sons, Inc.