Growth and isotopic labelling of adherent cells in small volumes of medium

Abstract

The in vivo labelling of viral or cellular components is usually conducted through the addition of radioactively labelled precursors to the culture medium. A limiting factor for isotope use is often the cost of isotope purchase and disposal. Therefore, significant savings can be achieved if the smallest possible volume of medium is employed. However, in the case of adherent cells growing in tissue culture dishes or multi-well plates, surface tension causes a very uneven distribution of the liquid due to the formation of a meniscus at the edge of the petri. This prevents the use of very small volumes of medium for cell growth and labelling because the cells at the center of the petri dish would dry out and die, especially after longer incubation periods. In this communication, we describe a technique whereby cells are grown in an area surrounded by a hydrophobic ring of Teflon, which greatly improves the distribution of the medium by eliminating the concave meniscus. This translates into a dramatic improvement in the condition of the cells, as well as the efficiency of labelling of phosphoproteins, such as the Simian Virus 40 large tumor antigen with 32P-orthophosphate or labelling of the cellular DNA with 3[H]thymidine. The technique is useful for any application where growth of cells in small volumes of medium is required.