Measurement of phagocytosis and of the phagosomal environment in polymorphonuclear phagocytes by flow cytometry.
Authors: Simons ER
Abstract
Phagocytes are the most important early components of the immune response, programmed to recognize, engulf, and destroy immune complexes (formed when antibodies recognize their specific antigens), foreign particles, bacteria, mycobacteria, apoptotic cells, etc. Neutrophils, monocytes, macrophages, and dendritic cells all participate in this process. Flow cytometry permits observation of phagocytes that have responded and, with the appropriate fluorescent probes, of the environment in the phagosome that has enclosed the foreign matter. This unit gives the background and the proto...
Source: Current Protocols in Cytometry - November 20, 2015 Category: Molecular Biology Tags: Curr Protoc Cytom Source Type: research
Life cycle analysis of unicellular algae.
Authors: Gerashchenko BI, Takahashi T, Kosaka T, Hosoya H
Abstract
Unicellular green alga is a very convenient object for flow cytometric characterization. Flow cytometry has been proposed as a quick and reliable tool for studying life cycle and growth of unicellular algae. Cell size of vegetating algae can be monitored in association with their DNA and endogenous chlorophyll content. Cells of interest (e.g., group of cells of a certain stage of the life cycle) in an asynchronously proliferating cell population can be sorted out for further microscopical or molecular biology studies. This methodological ap...
Source: Current Protocols in Cytometry - November 20, 2015 Category: Molecular Biology Tags: Curr Protoc Cytom Source Type: research
3D deconvolution microscopy.
Authors: Biggs DS
Abstract
3D deconvolution microscopy is a combination of optical and computational techniques that are used to maximize the observed resolution and signal from a biological specimen. Mathematical models are used to predict the distribution of out-of-focus light caused by the inherent optical limitations of the instrument, which can then be compensated for using computer algorithms. This unit will review the theory of image formation and characteristics of the point spread function (PSF) based on the instrument modality and objective lens parameters. A variety of commonly used deblurring a...
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
Click-iT proliferation assay with improved DNA histograms.
Authors: Krishan A, Hamelik RM
Abstract
The Click-iT EdU cell proliferation assay (Invitrogen) for detection of replicating cells is based on incorporation of EdU into newly synthesized DNA and its recognition by azide dyes via a copper mediated "click" reaction. In the protocol provided by Invitrogen, cells are fixed with paraformaldehyde and stained with 7-aminoactinomycin D (7-AAD) for DNA content analysis. Both of these procedures result in DNA histograms with a broad coefficient of variation. We have modified this protocol and show that after EdU incorporation, nuclei isolated by hypotonic lysis of ce...
Source: Current Protocols in Cytometry - November 20, 2015 Category: Molecular Biology Tags: Curr Protoc Cytom Source Type: research
Yeast cell cycle analysis: combining DNA staining with cell and nuclear morphology.
Authors: Calvert ME, Lannigan J
Abstract
In studies of eukaryotic cell cycle regulation, the budding yeast Saccharoymyces cerevisiae offers many advantages as a model system. Due to its simple growth requirements and genetic tractability, this organism is a powerful tool for investigating the molecular regulation of cell cycle control. One earlier disadvantage to performing cell cycle analyses in yeast was that existing methods were restricted to either visual analysis or flow cytometry, both of which present limitations in the scope and accuracy of the data obtained. This unit demonstrates the combined us...
Source: Current Protocols in Cytometry - November 20, 2015 Category: Molecular Biology Tags: Curr Protoc Cytom Source Type: research
Identification of endothelial cells and progenitor cell subsets in human peripheral blood.
Authors: Estes ML, Mund JA, Ingram DA, Case J
Abstract
An assay for circulating cell subsets in human peripheral blood by flow cytometry is used as a biomarker to determine cardiovascular disease risk and tumor responsiveness to chemotherapy since endothelial progenitor cells (EPCs) function in vasculogenesis and angiogenesis. Despite analytical advances in polychromatic flow cytometry (PFC), conventional approaches are routinely utilized to enumerate and isolate EPCs, which has led to varied results in clinical studies, potential cellular misidentification, and thus a lack of a plausible biological explan...
Source: Current Protocols in Cytometry - November 20, 2015 Category: Molecular Biology Tags: Curr Protoc Cytom Source Type: research
Web-based analysis and publication of flow cytometry experiments.
Authors: Kotecha N, Krutzik PO, Irish JM
Abstract
Cytobank is a Web-based application for storage, analysis, and sharing of flow cytometry experiments. Researchers use a Web browser to log in and use a wide range of tools developed for basic and advanced flow cytometry. In addition to providing access to standard cytometry tools from any computer, Cytobank creates a platform and community for developing new analysis and publication tools. Figure layouts created on Cytobank are designed to allow transparent access to the underlying experiment annotation and data processing steps. Since all flow cytometry fi...
Source: Current Protocols in Cytometry - November 20, 2015 Category: Molecular Biology Tags: Curr Protoc Cytom Source Type: research
Approaches to spectral imaging hardware.
Authors: Lerner JM, Gat N, Wachman E
Abstract
Instruments used for spectral, multispectral, and hyperspectral imaging in the biosciences have evolved significantly over the last 15 years. However, very few are calibrated and have had their performance validated. Now that spectral imaging systems are appearing in clinics and pathology laboratories, there is a growing need for calibration and validation according to universal standards. In addition, some systems produce spectral artifacts that, at the very least, challenge data integrity if left unrecognized. This unit includes a comparison of the band-pass ...
Source: Current Protocols in Cytometry - November 20, 2015 Category: Molecular Biology Tags: Curr Protoc Cytom Source Type: research
Amine-reactive dyes for dead cell discrimination in fixed samples.
Authors: Perfetto SP, Chattopadhyay PK, Lamoreaux L, Nguyen R, Ambrozak D, Koup RA, Roederer M
Abstract
Amine-reactive dyes, also known as LIVE/DEAD fixable dead cell stains, are a class of viability dyes suitable for identifying dead cells in samples that will be fixed. These dyes cross the cell membranes of dead cells, and react with free amines in the cytoplasm. Live cells exclude these dyes because their cell membranes are intact, and free dye is washed away after staining. Notably, the reaction is irreversible; therefore, when cells are fixed and permeabilized (as with intracellular staining procedure...
Source: Current Protocols in Cytometry - November 20, 2015 Category: Molecular Biology Tags: Curr Protoc Cytom Source Type: research
Detection of intracellular glutathione using ThiolTracker violet stain and fluorescence microscopy.
Authors: Mandavilli BS, Janes MS
Abstract
Glutathione plays an important role in protecting mammalian cells from oxidative stress and cell death. Because reduced glutathione (GSH) represents the large majority of intracellular free thiols, cell-permeant, thiol-reactive fluorescent probes represent potentially useful indicators of intracellular GSH. The ThiolTracker Violet stain (a registered trademark of Invitrogen) is a bright fluorescent probe that is highly reactive to thiols and can be used as a convenient and effective indicator of intracellular GSH and general redox status by a variety of detection m...
Source: Current Protocols in Cytometry - November 20, 2015 Category: Molecular Biology Tags: Curr Protoc Cytom Source Type: research
From image to data using common image-processing techniques.
Authors: Sysko LR, Davis MA
Abstract
A digital microscopy image is an array of number values, which with adequate contrast can be interpreted as spatial information. Through processing and analysis by mathematical means, using computer-assisted imaging software programs, raw image data contrast can be enhanced to improve the extraction of image features for measurement and analysis. This mathematical feature extraction (referred to as segmentation) provides the basis for general image processing. The methods discussed in this unit address common image analysis challenges such as object counting with touchi...
Source: Current Protocols in Cytometry - November 20, 2015 Category: Molecular Biology Tags: Curr Protoc Cytom Source Type: research
Microsphere-based flow cytometry protease assays for use in protease activity detection and high-throughput screening.
Authors: Saunders MJ, Edwards BS, Zhu J, Sklar LA, Graves SW
Abstract
This protocol describes microsphere-based protease assays for use in flow cytometry and high-throughput screening. This platform measures a loss of fluorescence from the surface of a microsphere due to the cleavage of an attached fluorescent protease substrate by a suitable protease enzyme. The assay format can be adapted to any site or protein-specific protease of interest and results can be measured in both real time and as endpoint fluorescence assays on a flow cytometer. Endpoint assays are easily adapted to microplate format for flo...
Source: Current Protocols in Cytometry - November 20, 2015 Category: Molecular Biology Tags: Curr Protoc Cytom Source Type: research
Evaluation and purchase of confocal microscopes: numerous factors to consider.
Authors: Zucker RM, Chua M
Abstract
The purchase of a confocal microscope is a difficult decision. Many factors need to be considered, which include hardware, software, company, support, service, and price. These issues are discussed to help guide the purchasing process.
PMID: 20938918 [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
Multi-photon imaging.
Authors: Padmanabhan K, Andrews SE, Fitzpatrick JA
Abstract
Multi-photon microscopy, now in its twentieth year, has developed into one of the most robust and powerful techniques for live cell and in vivo fluorescence imaging. Although its theoretical framework is nearly a century old, it has only become a practical tool for biological research with the development of ultrafast lasers and scanning microscopy techniques. In this unit, we outline the basic principles of multi-photon microscopy, paying special attention to technical considerations for biological applications. Furthermore, we discuss some commo...
Source: Current Protocols in Cytometry - November 20, 2015 Category: Molecular Biology Tags: Curr Protoc Cytom Source Type: research
