A review of reagents for fluorescence microscopy of cellular compartments and structures, Part II: reagents for non-vesicular organelles.
Authors: Kilgore JA, Dolman NJ, Davidson MW
Abstract
A wide range of fluorescent dyes and reagents exist for labeling organelles in live and fixed cells. Choosing between them can sometimes be confusing, and optimization for many of them can be challenging. Presented here is a discussion on the commercially-available reagents that have shown the most promise for each organelle of interest, including endoplasmic reticulum/nuclear membrane, Golgi apparatus, mitochondria, nucleoli, and nuclei, with an emphasis on localization of these structures for microscopy. Included is a featured reagent for each structur...
Source: Current Protocols in Cytometry - November 20, 2015 Category: Molecular Biology Tags: Curr Protoc Cytom Source Type: research
Flow cytometry-based cytotoxicity and antibody binding assay.
Authors: Alheim M
Abstract
Human leukocyte antigen (HLA) antibodies with the ability to activate complement are associated with an increased risk of early antibody-mediated graft rejection in kidney transplantation (KTx). Detection of these potentially harmful complement-fixing HLA antibodies is commonly performed via the complement-dependent cytotoxicity (CDC) assay according to protocols that were developed as early as 40 years ago. The read-out for this assay is based on manual scoring by visual inspection of cells under a fluorescence microscope. CDC is often used in combination with the flow cytometry...
Source: Current Protocols in Cytometry - November 20, 2015 Category: Molecular Biology Tags: Curr Protoc Cytom Source Type: research
Multiparameter analysis of apoptosis using lab-on-a-chip flow cytometry.
Authors: Wlodkowic D, Skommer J, Akagi J, Fujimura Y, Takeda K
Abstract
The age of microfluidic flow cytometry (µFCM) is fast becoming a reality. One of the most exciting applications of miniaturized chip-based cytometers is multivariate analysis using sampling volumes as small as 10 µl while matching the multiparameter data collection of conventional flow cytometers. We outline several innovative protocols for analyzing caspase-dependent cell death and cell cycle (DNA-content) profile using a fully integrated microfluidic flow cytometry system, Fishman-R. The first protocol describes the use of a new pl...
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 III: reagents for actin, tubulin, cellular membranes, and whole cell and cytoplasm.
Authors: Kilgore JA, Dolman NJ, Davidson MW
Abstract
Non-antibody commercial fluorescent reagents for imaging of cytoskeletal structures have been limited primarily to tubulin and actin, with the main factor in choice based mainly on whether cells are live or fixed and permeabilized. A wider range of options exist for cell membrane dyes, and the choice of reagent primarily depends on the preferred localization in the cell (i.e., all membranes or only the plasma membrane) and usage (i.e., whether the protocol involves fixation and permeabilization). For whole-cell or cytoplasmic imaging, the choice of reage...
Source: Current Protocols in Cytometry - November 20, 2015 Category: Molecular Biology Tags: Curr Protoc Cytom Source Type: research
How to build a time-gated luminescence microscope.
Authors: Jin D, Lu Y, Leif RC, Yang S, Rajendran M, Miller LW
Abstract
The sensitivity of filter-based fluorescence microscopy techniques is limited by autofluorescence background. Time-gated detection is a practical way to suppress autofluorescence, enabling higher contrast and improved sensitivity. In the past few years, three groups of authors have demonstrated independent approaches to build robust versions of time-gated luminescence microscopes. Three detailed, step-by-step protocols are provided here for modifying standard fluorescent microscopes to permit imaging time-gated luminescence.
PMI...
Source: Current Protocols in Cytometry - November 20, 2015 Category: Molecular Biology Tags: Curr Protoc Cytom Source Type: research
Real-time detection of protein trafficking with high-throughput flow cytometry (HTFC) and fluorogen-activating protein (FAP) base biosensor.
Authors: Wu Y, Tapia PH, Jarvik J, Waggoner AS, Sklar LA
Abstract
We combined fluorogen-activating protein (FAP) technology with high-throughput flow cytometry to detect real-time protein trafficking to and from the plasma membrane in living cells. The hybrid platform allows drug discovery for trafficking receptors, such as G protein-coupled receptors, receptor tyrosine kinases, and ion channels, which were previously not suitable for high-throughput screening by flow cytometry. The system has been validated using the β2-adrenergic receptor (β2AR) system and extended to other GPCRs. When a chemical libra...
Source: Current Protocols in Cytometry - November 20, 2015 Category: Molecular Biology Tags: Curr Protoc Cytom Source Type: research
OpenSource lab-on-a-chip physiometer for accelerated zebrafish embryo biotests.
Authors: Akagi J, Hall CJ, Crosier KE, Cooper JM, Crosier PS, Wlodkowic D
Abstract
Zebrafish (Danio rerio) embryo assays have recently come into the spotlight as convenient experimental models in both biomedicine and ecotoxicology. As a small aquatic model organism, zebrafish embryo assays allow for rapid physiological, embryo-, and genotoxic tests of drugs and environmental toxins that can be simply dissolved in water. This protocol describes prototyping and application of an innovative, miniaturized, and polymeric chip-based device capable of immobilizing a large number of living fish embryos for real-ti...
Source: Current Protocols in Cytometry - November 20, 2015 Category: Molecular Biology Tags: Curr Protoc Cytom Source Type: research
Intensity calibration and flat-field correction for fluorescence microscopes.
Authors: Model M
Abstract
Standardization in fluorescence microscopy involves calibration of intensity in reproducible units and correction for spatial nonuniformity of illumination (flat-field or shading correction). Both goals can be achieved using concentrated solutions of fluorescent dyes. When a drop of a highly concentrated fluorescent dye is placed between a slide and a coverslip it produces a spatially uniform field, resistant to photobleaching and with reproducible quantum yield; it can be used as a brightness standard for wide-field and confocal microscopes. For wide-field microscopes, calibratio...
Source: Current Protocols in Cytometry - November 20, 2015 Category: Molecular Biology Tags: Curr Protoc Cytom Source Type: research
A rapid and sensitive automated image-based approach for in vitro and in vivo characterization of cell morphology and quantification of cell number and neurite architecture.
Authors: Tapias V, Greenamyre JT
Abstract
Stereological methods for tissue cell counting, specifically for neuron quantification, decrease systematic error and sampling bias; however, they are tedious, labor intensive, and time consuming. Approaches for cell (neuron) quantification in vitro are not accurate, sensitive, or subsequently reproducible. Neuronal phenotype is related to alterations in cell morphology and neurite pattern. The techniques currently available for quantification of these features present several limitations. In this unit, we provide validated automated procedures for in vivo and in v...
Source: Current Protocols in Cytometry - November 20, 2015 Category: Molecular Biology Tags: Curr Protoc Cytom Source Type: research
Measurement of autophagy by flow cytometry.
Authors: Warnes G
Abstract
In recent years, flow cytometry has been used to detect the presence of autophagy mainly by the fluorescent antibody labeling of the autophagy marker, the microtubule associated protein LC3-II. Here we describe the indirect antibody labeling of LC3-II in cells displaying drug-induced autophagy by the use of rapamycin and chloroquine, as well as cells undergoing serum starvation. Although the mechanism of action of LysoTracker dyes is not fully understood, lysosomal mass increases during the autophagic process to enable the cell to produce autolysosomes. Given that LC3-II and Lyso...
Source: Current Protocols in Cytometry - November 20, 2015 Category: Molecular Biology Tags: Curr Protoc Cytom Source Type: research
Immunophenotyping of paucicellular samples.
Authors: Stacchini A, Demurtas A, Aliberti S
Abstract
Immunophenotyping of paucicellular samples may represent a diagnostic challenge in the flow cytometry (FC) laboratory routine, as the scarcity of cells limits the number of tests that can be performed. Specimens such as fine needle aspirates (FNA), human body fluids (BF), cerebrospinal fluid (CSF), or ocular fluid (OF) sent for FC investigations in the case of suspicion of lymphoma, or for lymphoma monitoring, may contain very low numbers of cells. In these cases, it is mandatory to obtain the largest amount possible of useful information from a single ...
Source: Current Protocols in Cytometry - November 20, 2015 Category: Molecular Biology Tags: Curr Protoc Cytom Source Type: research
Imaging autophagy.
Authors: Karanasios E, Stapleton E, Manifava M, Ktistakis NT
Abstract
Autophagy is a membrane-trafficking pathway activated to deliver cytosolic material for degradation to lysosomes through a novel membrane compartment, the autophagosome. Fluorescence microscopy is the most common method used to visualize proteins inside cells, and it is widely used in the autophagy field. To distinguish it from the cellular background, the protein of interest (POI) is either fused with a genetically encoded fluorescent protein or stained with an antibody that is conjugated to an inorganic fluorescent compound. Genetic ta...
Source: Current Protocols in Cytometry - November 20, 2015 Category: Molecular Biology Tags: Curr Protoc Cytom Source Type: research
The application of KillerRed for acute protein inactivation in living cells.
Authors: Jarvela TS, Linstedt AD
Abstract
Generating loss of protein function is a powerful investigatory tool particularly if carried out on a physiologically relevant timescale in a live-cell fluorescent imaging experiment. KillerRed mediated chromophore assisted light inactivation (CALI) uses genetic encoding for specificity and light for acute inactivation that can also be spatially restricted. This unit provides protocols for setting up and carrying out properly controlled KillerRed experiments during live-cell imaging of cultured cells.
PMID: 24984963 [PubMed - indexed for MEDLINE] (Source: C...
Source: Current Protocols in Cytometry - November 20, 2015 Category: Molecular Biology Tags: Curr Protoc Cytom Source Type: research
Application of the PrimRglo assay chemistry to multiplexed bead assays.
Authors: Fang L, Weis A, Wong LK, Yeh DC, Lai R, Corrie S, Barnard RT
Abstract
In this unit, we describe a multiplex microsphere quantitative PCR. The system is based on the use of two additional oligonucleotides within a single tube PCR reaction. The first oligonucleotide is modified with a single base pair mismatch and is otherwise equivalent to a universal sequence added to the forward PCR primer. Further, this first extra oligonucleotide is coupled to Luminex microspheres. The second additional oligonucleotide is designed to be complementary to the universal sequence, and is modified with the fluoresce...
Source: Current Protocols in Cytometry - November 20, 2015 Category: Molecular Biology Tags: Curr Protoc Cytom Source Type: research
Application of flow-FISH for dynamic measurement of telomere length in cell division.
Authors: Borisov VI, Korolkova OY, Kozhevnikov VS
Abstract
This method makes it possible to measure the fluorescence of a DNA probe in cells with known division number and targeted surface antigen. In fact, this method is a combination or consistent application of three other methods: cell tracking by vital dye, surface immunophenotyping, and flow-FISH. The idea in developing this method was to study telomere length changes in cells with known surface antigen after every new cell division. First, the in vitro cell culturing and staining with CFSE vital dye are performed. Then, cells are stained with surfac...
Source: Current Protocols in Cytometry - November 20, 2015 Category: Molecular Biology Tags: Curr Protoc Cytom Source Type: research
