Subcellular analysis culture

Many complex cellular processes are best smdied through the use of an appropriate cell model. Cell cultures provide a relatively stable and virtually unlimited supply of organelles for subcellular analysis. We will discuss organelle preparations that are commonly used in the author s laboratory... 

Fig. 1.2 Protein blot analysis of human therapeutic protease inhibitor (HTPI) produced in alfalfa cell cultures using different promoters and subcellular targeting peptides as shown. Equal amounts of total soluble proteins from cell cultures were separated by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) and blotted onto a polyvinyldifluoride (PVDF) membrane. Monoclonal anti-HTPI IgGs were used for detection.

In this chapter, we provide protocols to determine the ability of a peptide to mediate DNA internalization in cultured human tumor cells. Fluorescence-assisted cell sorting (FACS) analysis is used to obtain quantitative data on the time and temperature dependence of macromolecular delivery. Confocal microscopy is used to study the subcellular localization in both fixed and live cells. Fluorescently labeled transferrin and dextran are used to label the clathrin-dependent (15) and the non-clathrin, non-caveolar (16) endocytic compartments, respectively. Expression of a caveolin-l-YFP fusion protein is used to label cell surface caveolae and intracellular caveosomes (17). Finally a protocol, for the overexpression of dominant-negative dynamin [GTPase deficient dynamin-2 containing the amino acid substitution K44A (18)] is provided to evaluate the dynamin dependence of the uptake mechanism. 

The point analysis mode is used specifically to determine the concentration of the diffusible elements at the cellular level in complex tissues, in cells in culture, and at the subcellular level in individual cells. Under these circumstances information is provided that is not available from other techniques and that complements the information on ionic activities commonly obtained using ion-selective electrodes or fluorescent dyes. In addition, mapping techniques allow the investigation of 2D spatial distributions of elements at the nanometer level in intracellular compartments, yielding information that again cannot be provided by other techniques. 

It is hoped that this chapter will provide the reader with some appreciation of the fact that through an understanding of developmental nutrition, it has become possible to culture certain embryos under controlled conditions. Thus, it is now possible to study embryos in a manner previously restricted to bacteria and cells in culture. Embryos in vitro, however, provide a major advantage over bacteria and cell cultures in that analysis can be carried from the organism to the cellular and subcellular levels in seeking mechanisms of regulation. In spite of the advances... 

Sialic acid is an important component of membrane glycoproteins, which has been described in several reviews (e.g. Cook and Stoddart 1973, Hughes 1976, Jeanloz and Codington 1976, Warren 1976, Glick and Flowers 1978, and many others). Analysis of-subcellular membrane fractions has demonstrated the presence of NeuSAc in all fractions of cultured cells and tissue extracts. 

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