Methods in Cell Science

Numerous methods have been devised to facilitate the introduction of exogenous compounds into cells. The technique of electroporation allows the direct physical transfer of numerous kinds of molecules into essentially any cell, but the major application has been for transfection of DNA and this emphasis is recapitulated here. However, the conditions for transfer of DNA or other macromolecules are sufficiently similar that the same protocol is followed regardless. In addition, as electroporation involves a mechanism distinct from that of most other methods of transfection, it has distinct advantages and disadvantages relative to other transfection techniques. This review is designed to allow one to simplify the processes of determining whether electroporation is appropriate to a given experimental design, to indicate how to minimize the disadvantages, and to simplify the requisite process of parameter optimization required to evaluate and apply electroporation to the system of choice. Practical aspects are highlighted but the theoretical bases are discussed when relevant for application of the technique.

A method is described whereby endothelial cells are treated with several cycles of osmium and thiocarbohydrazide then critical point dried. Cells grown on Formvar-coated gold grids can be examined directly by transmission electron microscopy, cells grown on glass strips are mounted on stubs for scanning electron microscopy and are scanned without further coating with gold or palladium.

We have developed a method for the growth of WI-38 cells in a serum-free medium. Basal medium MCDB-104 is supplemented with platelet-derived growth factor (30 µg/ml), epidermal growth factor (100 ng/ml), insulin (5 µg/ml), transferrin (5 µg/ml), and dexamethasone (55 ng/ml). With this medium cells will grow at a rate and to an extent similar to that produced by medium containing 10% serum. During one growth cycle the cultures can accomplish up to seven population doublings.

Staurosporine (SSP) is a microbial alkaloid isolated from cultures of Streptomyces. For its ability to specifically inhibit protein kinase C and other serine/threonine and tyrosine protein kinases, SSP has been utilized recently to synchronize cells in G1 and/or G2 phases of the cell cycle. In the present paper we focus on the synchronizing properties of SSP with respect to three human normal cell types (PHA-activated peripheral blood lymphocytes, HS-68 and WI-38) and eight human tumor cell cultures (SV-40 transformed WI-38, SW48, SW480, A253, T-24, A549, MOLT-4 and HL-60). We describe procedures for maintenance and synchronization of these cells in culture. We provide the DNA/RNA flow cytometric methodology to verify cell synchronization and to evaluate the position of cell accumulation in specific phase and the level of synchrony. Synchronization in G1 is being achieved in the three normal cell lines after 24h SSP treatment with low concentrations (5–10 ng/ml) and in G2 after 24h treatment at higher SSP concentrations (50–100 ng/ml) in four of the eight tumor cell lines (A253, SW48, SW480 and A549). The other four tumor cell cultures (SV-40, WI-38, T-24, MOLT-4 and HL-60) show, at 50–100 ng/ml SSP concentration, an apparent G2 block, which is actually due to the presence of cells entering higher DNA ploidy levels. All other cell type/SSP dose combinations fail to induce cell synchronization. We also report a summary of the literature data about cell synchronization with SSP in other cell lines. Our results, together with the results from the literature, suggest that, while SSP may be useful for synchronizing normal cells in G1, its application for synchronizing tumor and/or transformed cells is limited. Critical comments follow, as well as suggestions and words of caution addressed to the future users of SSP as a cell synchronizing agent.