Pamapimod

THP-1 cells, growing in log phase, were collected by centrifugation and resuspended in RPMI 1640 containing 5.5×10^-5 M 2-mercaptoethanol and 10% fetal bovine serum to a final cell concentration of 2.5×10⁶ cells/ml. Dilutions of pamapimod were predispensed in 25 μl aliquots (before addition of cells) into round-bottom 96-well plates. The starting concentration was 100 μM in 5% dimethyl sulfoxide, and six half-log serial dilutions were made. After the addition of 200 μl of cell suspension and 25 μl of 5 μg/ml LPS in medium, the final dimethyl sulfoxide concentration was 0.5%. Compounds were diluted an additional 10-fold, and the final LPS concentration was 500 ng/ml. The cell suspensions and compound dilutions were combined and incubated for 30 min at 37°C in a 5% CO₂ humidified atmosphere, before the addition of LPS (or medium for non-LPS control samples). After the addition of LPS, plates were incubated for 2 h, followed by centrifugation to pellet cells. Cell supernatants were stored at 4°C until analysis for TNF-α content. TNF-α levels were determined by ELISA. Cytokine concentrations were determined.