Fluorescence microscopy fixed cells

Since our backbone 2 aPNA incorporates six Lys residues in its peptide sequence and is cationic at a physiological pH, we were optimistic that this aPNA would be taken up into cells without the need for any external carrier system. To answer the simple question of whether b2 aPNAs are intemahzed, a standard fluorescence microscopy experiment was performed to see if whole cells that were incubated with a fluorescent-labeled aPNA would internahze labeled material [70]. Chinese Hamster Ovary (CHO) cells in culture were incubated with BODIPY-la-beled TCCCT(b2) at 37 °C for various periods of time. Following incubation, the cells were rinsed in phosphate-buffered sahne (PBS), fixed with 4% formaldehyde at ambient temperature for 20 min, then washed with PBS and stored in a refrigerator until examined by fluorescence microscopy. 

Measuring FRET by fluorescence lifetime imaging microscopy (FRET-FLIM) offers the ability to see beyond the resolution of the optical system ( 10-100 times that of modern far field microscopes [5]). FRET efficiency can be used as a proxy for molecular distance, thereby allowing the easy detection and somewhat more challenging quantification of molecular interactions. Although many types of assay exist, FRET-FLIM is a highly suitable technique that is capable of in situ measurements of molecular interactions and conformation in living and fixed cells. 

Figure 11.2 Subcellular distribution of fluorescein-labeled DNA fragments and plasmid DNA following microinjection into the cytoplasm. Double stranded circular plasmid DNA (3 kb and 6 kb) and DNA fragments (20,100,250 and 1 kb) were covalently labeled with fluorescein and microinjected into the cytoplasm of adherent HeLa cells as described in Lukacs et al., 2000. Following micro injection, cells were either fixed or incubated for 45 minutes at 37 °C and the distribution of DNA was visualized by fluorescence microscopy, (see Color Plate 10)...

Currently, fluorescence microscopy is one of the most useful techniques for the examination of drug localization in fixed and live cells. However, to use this technique, the drug must be fluorescent, or labeled with a fluorescent tag. 

Fluorescence Microscopy Can Localize and Quantify Specific Molecules in Fixed and Live Cells... 

In immunofluorescence microscopy, specific proteins and organelles in fixed cells are stained with fluorescence-labeled monoclonal antibodies. Multiple proteins can be localized in the same sample by staining with antibodies labeled with different fluorochromes. 

Asymmetric localization of intracellular proteins and signals directs movement dimng axon guidance, endothelial cell invasion, and immune cell migration. In these processes, cell movement is guided by external chemical cues in a process known as chemotaxis. In particular, leukocyte migration in the innate immune system has been studied in the human neutrophil-like cell line (HL-60). Here, we describe the maintenance and transfection of HL-60 cells and explain how to analyze their behavior with two standard chemotactic assays. Finally, we demonstrate how to fix and stain the actin cytoskeleton of polarized cells for fluorescent microscopy imaging. 

When cells are exposed to doses of a water-soluble form of actinomycin D, Act D-mannitol (Sigma), at concentrations of O.l-l.O /i.g/ml (made from a 1 mg/ ml stock solution dissolved in distilled water) for times of 1-4 hr it is possible to obtain segregated nucleoli with fibrillar caps separated from granular regions (Fig. 7a). These optimally segregated nucleoli can then be fixed and prepared for fluorescence microscopy or immunoelectron microscopy in order to localize... 

Triton X-100). Alternatively, cells can be fixed and permeablized with organic solvents such as methanol and acetone. The type of fixative depends on the cellular components that need to be detected and the employed antibodies. Cellular components are then detected either directly by means of dyes (e.g., fluorescent dyes that bind to and stain DNA) or antibodies that are labeled with fluorophores. Alternatively, an indirect detection can be performed by means of primary antibody that specifically binds to a protein of interest and a secondary antibody that recognizes the primary antibody and is labeled for detection (usually with a fluorophore). Finally, samples are analyzed by means of fluorescence microscopy. 

Fig. 4 Metabolic DNA labeling using alkyne- or alkene-modified nucleosides. A 5-Ethynyl-2 -deoxyuridine (EdU) [153], (2 S)-2 -deoxy-2 -fluoro-5-ethynyluridine (F-ara-EdU) [160] and 5-vinyl-2 -deoxyuridine (VdU) [167], B Labeling strategy cells are incubated with the alkyne-modified nucleoside EdU, which is incorporated into the cellular genome in proliferating cells. Subsequently, the cells are fixed, permeabilized and components for the Cu -mediated azide alkyne cUck reaction with flumescent azides are added. Detection can be carried out by flow cytometry or fluorescence microscopy...

Immunofluorescence staining permits detection of protein antigens in situ, in order to investigate the subcellular localization or cellular distribution within a tissue. The cells or tissue sections are fixed and incubated with the specific primary monoclonal antibody. The antigen-primaiy monoclonal antibody complex is bound by a second antibody conjugated to a fluorescent dye, such as rhodamine-p-isothiocyanate or fluorescein isothiocyanate, for detection by fluorescence microscopy. Immunofluorescence staining is described in more detail in Chapter 16. 

Immrmolabelling of organelles or structures by fluorescent antibodies in living cells has the advantage, compared to work in fixed cells, that the dynamics of the labelled structures or molecules can be studied by for example time-lapse microscopy. Also, labelling in living cells does not suffer from possible problems induced by the fixation or permeabilization of the cells as is necessary for immimofluorescence or electron microscopy. 

Fig. 6 A rhenium tricarbonyl complex conjugated to a bombesin analogue and an inter-calator, compound 7, visualised in fixed PC-3 cells by fluorescence microscopy (left), where green represents the complex and blue the DAPI nuclear stain.

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