Single fixed cell

Visual methods have been virtually displaced for most determinations by methods depending upon the use of photoelectric cells (filter photometers or absorptiometers, and spectrophotometers), thus leading to reduction of the experimental errors of colorimetric determinations. The so-called photoelectric colorimeter is a comparatively inexpensive instrument, and should be available in every laboratory. The use of spectrophotometers has enabled determinations to be extended into the ultraviolet region of the spectrum, whilst the use of chart recorders means that the analyst is not limited to working at a single fixed wavelength. 

FIGURE 15.10 Landscape image of a two-dimensional capillary electrophoresis analysis of the protein content of a single fixed MCF-7 breast-cancer cell. The data consist of a few high-amplitude components. 

The measurements can be made at various levels from that of subcellular organelles, to single cells, cell populations and even tissues. They can be made on either fixed cells or on live cells that are incubated under physiological conditions. They can be made in end point assays or kinetically, in real time. Compared to previous manual methods, automation provides a marked improvement in the capacity for sample and experiment throughput, the precision of measurement and in the sheer number and diversity of parameters measureable for an experiment. Consolidation of the technical capabilities allo vs unparalleled vhthin-experiment, cross-comparisons of biochemical, morphological and functional parameters. Compared to flo v cytometry it offers substantially greater analytic capability for morphometric and kinetic parameters, although for substantially lo ver numbers of cells. 

Fig. 18.2. Raman spectroscopy of live, fixed and dried cells. Raman spectrum of a single cell construct provides a unique biochemical fingerprint , which provides a snap shot of the entire biomolecular components. The mean Raman spectra (4 separate measurements) of a single live, fixed and desiccated epithelial cell are compared (a). Fixation and desiccation influence cellular biochemistry. Desiccation distorts Raman bands describing all cellular biopolymers, especially proteins. Distinct biochemical changes in the secondary structure of proteins in the fixed cell can also be detected. Similar results were obtained in several other cells when analysed under similar conditions. Light microscope pictures of the cells in live cell culture (b), and after fixation (c) and desiccation (d) are shown. Scalebar = 10 pm. [3]...

The fatty acid components of a phospholipid may vary, and thus phosphatidyl serine, as well as most other phospholipids, represents a class of molecules rather than a single species. As a result, a single mammalian cell may contain thousands of distinct phospholipids. Phosphatidyl inositol is unusual in that it has a nearly fixed fatty acid composition. Stearic acid usually occupies the C-1 position and arachidonic acid (Section 22.6.2) the C-2 position. 

Cells are often fixed and destroyed so that intact single living cells cannot be analyzed. These restrictions make current assays of apoptosis cumbersome and impractical for clinical use. 

Here, we describe the details of an improved cDNA subtraction method based on a cDNA subtraction approach previously applied to single cells (6), which in turn is based on a global cDNA amplification protocol (PolyAPCR) used to produce amplified cDNAs, representing all the mRNAs present in the samples as small as a single cell (7,8). PolyAPCR has been applied successfully to a wide range of samples, including single micromanipulated cells (6,7,9,10), antibody fractionated populations (11), and fixed tissues (12). 

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