69] Methods for enucleation and reconstruction of interferon-producing cells

Abstract

This chapter describes the methods for enucleation and reconstruction of interferon-producing cells. The extent of enucleation can be increased by centrifugation in the presence of the drug, probably because the nucleus is forced into a cytoplasmic stalk, which subsequently severs spontaneously. The percentage of efficiency of enucleation is readily estimated by staining the cytoplasts with crystal violet after recovery; any nucleated cells are then obvious, and both nucleated and enucleated cells can be counted. Cell reconstruction can be separated into two stages—namely, the agglutination of cytoplasts and karyoplasts by the Sendai virus and the actual fusion. Mouse L cells produce high titers of interferon (up to 10 5 international research units) when induced with NDV strain F. However, enucleated cells do not produce any interferon, whereas reconstructed cells produce as much interferon per cell as do normal L cells. Thus the interferon system, requiring a nuclear gene and cytoplasmic protein synthesis, has also been reconstructed.