Fig. 1.

Protocol employed for secretome analysis of adjuvant-stimulated primary human newborn and adult monocytes. Human monocytes, isolated as described in Methods, were cultured under serum-free conditions for 18 h in the presence of sterile buffer (control), Alum [5 μg/ml], MPLA [100 ng/ml], and R848 [5 μm]. Supernatant proteins were precipitated prior to SDS-PAGE and trypsin digestion. The resulting peptide mixtures were subjected to LC-MS/MS on a QE mass spectrometer (3 h gradient). Several levels of data analysis were undertaken for protein identification and secretome comparison.