This chapter describes a method, combining the actions of cytochalasin B (CB) and high g forces, in order to obatin 99% enucleation. CB is used for preparing anucleate cytoplasms of the human fetal diploid cell strain WI-38 for cell fusion studies. Special Lucite plates that withstand high g forces can be used as a substrate for the cells. Because WI-38 cells do not attach well to Lucite surfaces, glass cover slips were glued with clear epoxy cement to one surface of the Lucite plates. It is important to mix the epoxy resin and hardener very thoroughly; otherwise the bond will contain many refracting interfaces that make examination of the cells difficult. Cytochalasin B is poorly soluble in water, and is thus, first dissolved in dimethyl sulfoxide (DMSO). Approximately, 25 ml of CB medium is added to each centrifuge tube containing one Lucite-cover slip plate. The tubes are balanced to within 0.1 gm of each other with additional CB medium. The Lucite-cover slip plates are then centrifuged in a Beckman SW 25.1 or SW 27 rotor for 30 minutes at various speeds. The enucleation of cells is highly temperature dependent between 0°C and 25°C with much greater efficiency at the higher temperatures.
ASJC Scopus subject areas
- Cell Biology