Preparation of Drosophila Kc cell nuclear extracts for in vitro splicing

This protocol describes how to prepare nuclear extracts from cultured Drosophila cells. It uses the Kc cell line, which grows without fetal bovine serum and so is suitable for large-scale suspension cultures. The suspension cells are harvested by centrifugation, washed to remove media components, and resuspended in a low-salt (hypotonic) buffer that makes the cells swell in size and easy to homogenize. Usually, nuclear extract preparations are performed on 4-32 L of cells at ∼ 3.5 × 10(6) cells/mL.