Analysis of protein and lipid dynamics using confocal fluorescence recovery after photobleaching (FRAP).
Authors: Day CA, Kraft LJ, Kang M, Kenworthy AK
Abstract
Fluorescence recovery after photobleaching (FRAP) is a powerful, versatile, and widely accessible tool to monitor molecular dynamics in living cells that can be performed using modern confocal microscopes. Although the basic principles of FRAP are simple, quantitative FRAP analysis requires careful experimental design, data collection, and analysis. In this unit, we discuss the theoretical basis for confocal FRAP, followed by step-by-step protocols for FRAP data acquisition using a laser-scanning confocal microscope for (1) measuring the diffusion of...
Source: Current Protocols in Cytometry - November 20, 2015 Category: Molecular Biology Tags: Curr Protoc Cytom Source Type: research
Spectral flow cytometry.
Authors: Nolan JP, Condello D
Abstract
Interest in measuring the complete fluorescence spectra of individual cells in flow can be traced to the earliest days of flow cytometry. Recent advances in detectors, optics, and computation have made it possible to make full spectral measurements in the sub-millisecond time frame in which flow cytometry measurements typically occur. This opens up new possibilities for applying spectroscopy to the analysis of individual cells. This unit reviews historical and contemporary approaches to spectral flow cytometry, as well as instrument design, calibration, and data analy...
Source: Current Protocols in Cytometry - November 20, 2015 Category: Molecular Biology Tags: Curr Protoc Cytom Source Type: research
Evaluation and purchase of an analytical flow cytometer: some of the numerous factors to consider.
Authors: Zucker RM, Fisher NC
Abstract
When purchasing a flow cytometer, the decision of which brand, model, specifications, and accessories may be challenging. The decisions should initially be guided by the specific applications intended for the instrument. However, many other factors need to be considered, which include hardware, software, quality assurance, support, service, and price and recommendations from colleagues. These issues are discussed to help guide the purchasing process.
PMID: 23292706 [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
Contrast enhancement in light microscopy.
Authors: Ernst Keller H, Watkins S
Abstract
The optical microscope is a fundamental component of an image cytometry system. This unit covers the basic concepts of light microscopy, including Köhler illumination, resolution, contrast, and numerical aperture, and reviews the many types of instruments and techniques for contrast enhancement.
PMID: 23292707 [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
Comparative and practical aspects of localization-based super-resolution imaging.
Authors: Laevsky GS, O'Connell CB
Abstract
Super-resolution microscopy overcomes diffraction to generate images with superior resolution compared to conventional light microscopy. Localization-based super-resolution methods result in up to ten-fold improvement in resolution by determining the positions of fluorescent molecules with sub-pixel accuracy. This process critically depends on controlled emission at the level of individual fluorophores so that fluorescence is non-overlapping, allowing for accurate centroid determination of diffraction-limited spots by Gaussian fitting of the pixel intensities. The...
Source: Current Protocols in Cytometry - November 20, 2015 Category: Molecular Biology Tags: Curr Protoc Cytom Source Type: research
Flow cytometry-based quantification of cell proliferation in the mixed cell co-culture.
Authors: Gerashchenko BI, Howell RW
Abstract
In cell communities, among the crucial signals that govern cell function are those generated locally by surrounding cells. A co-culture of mixed homotypic or heterotypic cells, which is often used in various fields of experimental biology and medicine, can be applied for elucidation of the role of cell proximity in modulating proliferative responses. Quick and reliable quantification of the changes in proliferation of each of the mixed cell populations as a result of their co-culture is of importance. For this purpose, flow cytometry together with fluorescent tr...
Source: Current Protocols in Cytometry - November 20, 2015 Category: Molecular Biology Tags: Curr Protoc Cytom Source Type: research
Time-lapse microscopy approaches to track cell cycle and lineage progression at the single-cell level.
The objective of this unit is to present the basic methodology for capturing time-lapse sequences and touch upon subsequent mining of the data for deriving event curves and possible cell lineage maps.
PMID: 23546776 [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
Flow cytometric analysis of cell division by dilution of CFSE and related dyes.
Authors: Lyons AB, Blake SJ, Doherty KV
Abstract
The technique described in this unit uses the intracellular fluorescent label carboxyfluorescein diacetate succinimidyl ester (CFSE) to track proliferating cells. Covalently bound CFSE is divided equally between daughter cells, allowing discrimination of successive rounds of cell division. The technique is applicable to in vitro cell division, as well as to in vivo division of adoptively transferred cells and can resolve eight or more successive generations. CFSE is long lived, permitting analysis for several months after cell transfer, and has the same spec...
Source: Current Protocols in Cytometry - November 20, 2015 Category: Molecular Biology Tags: Curr Protoc Cytom Source Type: research
Assessment of cell viability.
Authors: Johnson S, Nguyen V, Coder D
Abstract
Cell viability may be judged by morphological changes or by changes in membrane permeability and/or physiological state inferred from the exclusion of certain dyes or the uptake and retention of others. This unit presents methods based on dye exclusion, esterase activity, and mitochondrial membrane potential, as well as protocols for determining the pre-fixation viability of fixed cells either before or after fixation with amine-reactive dyes suitable for a range of excitation wavelengths. Membrane-impermeable dead cell and live cell dyes as well as dye-exclus...
Source: Current Protocols in Cytometry - November 20, 2015 Category: Molecular Biology Tags: Curr Protoc Cytom Source Type: research
Flow cytometry of the side population (SP).
Authors: Petriz J
Abstract
The side population (SP) has become an important hallmark for the definition of the stem-cell compartment, especially for the detection of stem cells and for their physical isolation by fluorescence-activated cell sorting (FACS). SP cells are CD34(-) and were discovered using ultraviolet excitation based on the efflux of Hoechst 33342 (Ho342). Although the method works as originally described, the protocol is difficult for most investigators to perform: first, because the ability to discriminate SP cells is based on the differential retention of Ho342 during a functional assay; s...
Source: Current Protocols in Cytometry - November 20, 2015 Category: Molecular Biology Tags: Curr Protoc Cytom Source Type: research
Fluidics.
Authors: Austin Suthanthiraraj PP, Graves SW
Abstract
The use of fluidics is implicit in a technology named "flow cytometry," which flows a cell or particle through a sensing volume to obtain serial analysis of particles on a one by one basis. This flow of particles enables flow cytometry to collect information on multiple particle populations, giving it a distinct advantage over bulk analysis approaches. Moreover, flow cytometers can analyze thousands of particles per second in a single flowing stream. Additionally, use of volumetric sample delivery makes it possible for flow cytometers to accurately coun...
Source: Current Protocols in Cytometry - November 20, 2015 Category: Molecular Biology Tags: Curr Protoc Cytom Source Type: research
Cytometry in malaria--a practical replacement for microscopy?
Authors: Shapiro HM, Apte SH, Chojnowski GM, Hänscheid T, Rebelo M, Grimberg BT
Abstract
Malaria, caused by protozoan Plasmodium parasites, kills ~800,000 people each year. Exact figures are uncertain because presumptive diagnoses are often made without identifying parasites in patients' blood either by microscopy, using Giemsa's century-old stain, or by simpler tests that are ultimately dependent on microscopy for quality control. Microscopy itself relies on trained observers' ability to detect subtle morphological features of parasitized red blood cells, only a few of which may be present on a slide. Qu...
Source: Current Protocols in Cytometry - November 20, 2015 Category: Molecular Biology Tags: Curr Protoc Cytom Source Type: research
A review of reagents for fluorescence microscopy of cellular compartments and structures, part I: BacMam labeling and reagents for vesicular structures.
Authors: Dolman NJ, Kilgore JA, Davidson MW
Abstract
Fluorescent labeling of vesicular structures in cultured cells, particularly for live cells, can be challenging for a number of reasons. The first challenge is to identify a reagent that will be specific enough where some structures have a number of potential reagents and others very few options. The emergence of BacMam constructs has allowed more easy-to-use choices. Presented here is a discussion of BacMam constructs as well as a review of commercially-available reagents for labeling vesicular structures in cells, including endosomes, peroxisomes, lyso...
Source: Current Protocols in Cytometry - November 20, 2015 Category: Molecular Biology Tags: Curr Protoc Cytom Source Type: research
Building a live cell microscope: what you need and how to do it.
Authors: Watkins SC, St Croix CM
Abstract
In this post-genomic era, the predominant focus of biomedical research has moved towards elucidating the functionality of molecules at the cellular and subcellular level in integrated living systems. As such, biologic imaging has moved beyond the collection of static "snapshots" of the cellular state to the real-time visualization of cellular and molecular behavior in three-dimensional (3D) space. Live-cell imaging techniques can be used to assess protein abundance, as well as determine the functional role(s) and interactions of multiple unique molecules concurrent...
Source: Current Protocols in Cytometry - November 20, 2015 Category: Molecular Biology Tags: Curr Protoc Cytom Source Type: research
Kinetic viability assays using DRAQ7 probe.
Authors: Wlodkowic D, Akagi J, Dobrucki J, Errington R, Smith PJ, Takeda K, Darzynkiewicz Z
Abstract
Cell death within cell populations is a stochastic process where cell-to-cell variation in temporal progression through the various stages of cell death arises from asynchrony of subtle fluctuations in the signaling pathways. Most cell death assays rely on detection of the specific marker of cell demise at the end-point of cell culturing. Such an approach cannot account for the asynchrony and the stochastic nature of cell response to the death-inducing signal. There is a need therefore for rapid and high-th...
Source: Current Protocols in Cytometry - November 20, 2015 Category: Molecular Biology Tags: Curr Protoc Cytom Source Type: research
