Cell fluorescence staining

Fluorescent Staining. Certain methods of tissue staining differentially detect the presence of particular chemical components in cells. One approach is to use a fluorescent label to trace components inside the cell. Fluorescent staining not only attests to the presence or absence of certain components in cells but also provides their precise localization in a particular cell or tissue type. For example, immunochem-istry uses fluorescent antibodies that specifically bind with the target structures in the tissue so that the labeled antibodies will highlight the presence of the structures of interest in the analyzed specimen. Some applications of these techniques are staining of virus-infected cells in tissues and detection of immune-complex deposition in the kidneys and skin of patients with lupus. 

It had been found that if bacteria are stained with acridine orange and examined under fluorescent microscopy, viable, as dishnct from dead, cells fluoresce with an orange-led hue. This basic observation has been adapted to an ingenious method of determining bacterial content and may be completed within 1 hour. 

During the past two decades, cell-biological and biomedical research has greatly benefited from innovations in fluorescence microscopy. Both the increase in the repertoire of fluorescence staining techniques at the (sub)cellular level and the development of a multitude of novel fluorescence microscopy techniques contributed significantly. 

Fluorescent stains such as DAPI and ethidium bromide bind to DNA (sometimes even mitochondrial DNA) and have many uses, including location of nuclei and rapid detection of microorganisms or virus formation (22). The lipophilic stain DiOC(6) can be used to stain mitochondria and endoplasmic reticulum in living cells (23,24), and is also a potential-sensitive stain. 

Plant Cells and Tissues Structure-Function Relationships. Methods for the Cytochemical/Histochemical Localization of Plant Cell/Tissue Chemicals. Methods in Light Microscope Radioautography. Some Fluorescence Microscopical Methods for Use with Algal, Fungal, and Plant Cells. Fluorescence Microscopy of Aniline Blue Stained Pistils. A Short Introduction to Immunocytochemistry and a Protocol for Immunovi-sualization of Proteins with Alkaline Phosphatase. The Fixation of Chemical Forms on Nitrocellulose Membranes. Dark-Field Microscopy and Its Application to Pollen Tube Culture. Computer-Assisted Microphotometry. Isolation and Characterization of... 

Some fluorescent DNA stains can also be used for chromosome counterstaining, for detection of hybridized metaphase or interphase chromosomes in fluorescence in situ hybridization assays or for identifying apoptotic cells in cell populations (http //probes.invitrogen.com/handbook/sections/0806.html). For instance, Vybrant Apoptosis Assay Kit 4 (Molecular Probes) detects apoptosis on the basis of changes that occur in the permeability of cell membranes. This kit contains ready-to-use solutions of both YO-PRO-1 and propidium iodide nucleic acid stains. YO-PRO-1 stain selectively passes through the plasma membranes of apoptotic cells and labels them with moderate green fluorescence. Necrotic cells are stained red-fluorescent with propidium iodide. 

Mitochondria are distinct organelles with two membranes. The outer membrane limits the organelle and the inner membrane is thrown into folds or shelves that project inward and are called cristae mitochondriales. The uptake of most mitochondrion-selective dyes is dependent on the mitochondrial membrane potential. Conventional fluorescent stains for mitochondria, such as rhodamine and tetramethylrosamine, are readily sequestered by functioning mitochondria. They are, however, subsequently washed out of the cells once the mitochondrion s membrane potential is lost. This characteristic limits their use in experiments in which cells must be treated with aldehyde-based fixatives or other agents that affect the energetic state of the mitochondria. To overcome this limitation, the research... 

Precise quantifications are an important quality in molecular biology. There are slight differences in the methods used for global and targeted proteomics. In experiments intended to visualize as many proteins as possible, it is highly desirable to have a parallel quantification method that builds on the display technique. For 2D gel electrophoresis, fluorescent staining methods are under development (Urwin and Jackson, 1993), but they still lack overall sensitivity. Labeling proteins with radioactive isotopes is the most precise method for quantification but is limited to cell cultures, and alternatives are desirable. Recently, a precise method... 

Fluorescence staining for flow cytometric analysis falls into three categories methods in which a fluorescent ligand accumulates on or within the cell (see Chapters 36,38) methods that require the ligand to interact with a cellular component to release the fluorophore or result in light emission (see Chapters 34,39) and methods that rely on fluorophore-coupled antibody binding (see Chapters 32,33). 

The actual response of monoclonal antibodies with individual cells is usually visualized either directly (typically using fluorescent stains) or indirectly [using the reaction of antibody labeled with horseradish peroxidase (HRP) or other enzymes] with diaminobenzidine (DAB) (or other substrate while using other enzymes) under the microscope or in the flow cytometer. The latter, however, is not employed routinely in CSF immunocytology, although it has an advantage in clinical hematology. 

Log amplifiers are usually employed to analyze fluorescence signals from cells with stained surface markers, because these cells often exhibit a great range of fluorescence intensities. Linear amplifiers are usually employed for analyzing the DNA content of cells, because the DNA content of cells does not normally vary by more than a factor of 2 (e.g., during cell division). Linear amplifiers may be used to analyze forward and side scatter signals, but practice here is apt to vary from lab to lab. With either linear or logarithmic amplification,... 

A layer-by-layer microfluidics technology was used to construct a 3D microscale hierarchical tissue-like structure. For instance, three layers of tissues, fibroblasts (human lung), myocytes (smooth muscle cells), and endothelial cells (human umbilical vein) were cultured on top of each other using consecutive microchannel cell matrix delivery. Cell viability was confirmed by fluorescent staining [862]. 

Fig. 8 (a) Nanopatteming by EBL A silcion substrate is functionalized with an amino-silane and coated with BSA. A focused electron beam is employed to write nanopattems into the BSA film. Proteins from solution can selectively adsorb into the nanopattems and guide the formation of cell-substrate contacts, (b) Fibroblast on fibronectin 10 x 10 nanodot matrix created by electron beam lithography. Cells spread, and fluorescent staining of intracellular proteins shows that focal contacts are located on the nanodots actin green), fibronectin red), and vinculin blue). Areas a, b, and c are shown magnified. (Figure in part reproduced with permission from [103])... 

Fig. 9.4. Fluorescence staining for mycoplasma. Cells were stained with Hoechst 33258 which fluoresces with DNA. (a) Vero cells infected with M. arginini which show marked extranuclear fluorescence (b) negative control cells. The photographs (x40) were kindly supplied by Dr. Rae of Moredun Animal Health Ltd.

Difficulty is encountered in detecting mycoplasma in certain cell lines and, for this reason, indicator cells e.g. mouse 3T3 cells are often used. 24 hours after establishing a cover slip culture of 3T3 cells a sample of the supernatant of the cells under test is added to the culture. Positive and negative controls should also be included. Three to four days later cultures are tested using fluorescence staining and another technique. 

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