One challenging point in analyzing cellular secretome collected as conditioned medium iscross-contamination by cell culture media components, especially bovine serum proteins.A common approach for serum removal is to wash the cells, an alternative is to growcells using serum-free conditions. Given that the sample processing may influence thephenotype of cells and thus the secretome, it is important to establish the optimal protocolfor each cell type. In this study, we compared two methods for preparing conditionedmedium from human adipocytes derived from mesenchymal stem cells. Cells were eitherwashed twice with PBS or cultured the last four days of differentiation in serum-freeadipogenic medium. Gene expression of the cells was evaluated by using real-time PCR and1D LC-MS/MS was used to compare secreted proteins present in the culture supernatants.Surprisingly, results showed significant differences in gene expression patterns of the cellsand in protein content of the conditioned media and suggested that PBS washes inducedsevere modifications of the phenotype of cells and thus changes in protein secretionprofiles. These data emphasize the significant variations in protein species related tocell manipulations and underline the importance of procedure optimization prior to anyproteomic investigation