Long-term hyperosmotic condition culture extends mouse macrophages half-life and alters p53, Bax, and Bcl-2 levels. BAL macrophages were obtained from 3 consecutive lavages of BALB/c mice lungs. Cells were cultivated in Dulbecco's minimal essential medium in the presence of 10 ng/ml M-CSF. Hyperosmolarity was obtained by adding up to 300 or 600 mOsm Mannitol to the culture medium. Cell viability (OD 450 nm) was assessed every day through an MTT test (A). p53 (B) and Bcl-2 (C) protein expression was measured every other day in whole protein extracts (OD 450 nm) as described in the Materials and Methods section. Rnase protection assay (RPA) of Bax and Bcl-2 was performed (D) (L32 is used as control). Dark triangles represent 300 mOsm cultures and light squares represent 600 mOsm cultures. Data were reproduced in three experiments, each composed of three independent macrophage samples. One representative experiment is presented and each point represents the average of the three samples.