Induction and identification of mast cells from long-term culture of mouse spleen cells without conditioned medium

Abstract

A simple method is described for the preparation of large numbers of mast cells from mouse spleen cells in vitro. Mouse spleen cells were cultured with RPMI 1640 medium supplemented with 10% FCS and 2-ME. Half of the total volume of the medium was changed every 4–5 days. Mast cell numbers increased with the culture time and reached a peak between 16 and 20 days. Using this method, 2 × 10 6 mast cells could be induced from 1 × 10 7 nucleated normal spleen cells. T cells and supernatant derived from ConA-stimulated T cells were unnecessary for mast cell induction. Phenotype analysis by FACS showed that Thy1,2, L3T4, Ly-2, Ig, B220, Asialo G M1, and WGA receptors were all negative but functional IgE receptors were positive. The granules in the cells could be stained by alcian blue but not by safranin. There was 1.632 ± 0.024 μg stored histamine in 1 × 10 6 of the cells. Histamine was released from the cells in an antigen-induced and IgE-mediated process. Compound 48/80 and A23187 induced degranulation of the cells, and the mast cells were able to respond to ConA.