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Development of strategies that allow for identification of secreted factors in conditioned media is of significance for a wide range of research areas. Secreted factors are involved in intercellular communication and might also be biomarkers of potential clinical importance, used for early detection and diagnosis of disease. The aim of this study was to investigate whether metabolic labeling combined with mass spectrometry could be used to identify secreted proteins in serum-containing conditioned medium. Earlier proteomic studies of conditioned media have been performed on cells cultured in serum-free media. In the present study the fact that only the proteins derived from the cells contain the incorporated isotopically labeled amino acid was taken advantage of, making it possible to differentiate released proteins from medium proteins. The second objective was to examine whether any quantitative differences in the secretion profiles between primary astrocytes and astrocytes in a scratch injury model of reactive astrogliosis could be found. To our knowledge, this is the first study to identify secreted proteins in serum-containing medium using a proteomic approach involving stable isotope labeling by amino acids in cell cultures and mass spectrometry.