Purification of cytoplasts (enucleated cells) and karyoplasts (nuclei) from mouse splenic lymphocytes with cytochalasin-B

Abstract

Murine spleen and lymphoma cells were almost completely enucleated in suspension by centrifugation in discontinuous Ficoll gradients in the presence of cytochalasin-B. Viability of cytoplasts (cells lacking nuclei) and karyoplasts (nuclei) was over 90%. Pure fractions of cytoplasts or karyoplasts were then obtained by fractionation of the cytochalasin-B treated cells with unit gravity sedimentation in bovine serum albumin. Protein synthesis was seen in cytoplasts when they were stimulated with mitogens, such as lipopolysaccharide (LPS). This method can consistently yield pure fractions of morphologically and functionally intact karyoplasts or cytoplasts from normal lymphocytes with very low contamination of nucleated cells. Pure karyoplasts or cytoplasts obtained in this way could be extremely useful for subcellular analysis of lymphocyte activation.