Differentiation and Cytokine Response of THP-1 Cells

Experiments in THP-1 cells were based on methods previously described and expanded upon (97 (link)). After growth to approximately 8 × 10⁵ cells/ml in C-RPMI, monocyte-like THP-1 cells were pelleted by centrifugation at 200 × g for 5 min and resuspended in C-RPMI to a concentration of 1 × 10⁶ cells/ml. PMA was then added to a final concentration of 50 nM, and the cells were seeded in 48-well tissue culture plates at 4 × 10⁵ cells/well. Commercial LPS (LPS-EB from E. coli O111:B4; InvivoGen) at 10 ng/ml, purified LOS at 10 ng/ml, or paraformaldehyde-treated bacteria at different multiplicities of infection (MOI) were added to the wells during this time for training. The cells were allowed to differentiate for 24 h into a macrophage-like phenotype before the medium was discarded and replaced with fresh C-RPMI, and they were rested for 16 h before challenge with C. jejuni HS:19 at an MOI of 5. After 24 h of challenge, the supernatants were removed and cytokine production measured using TNF (BD-Biosciences), IL-6 (BD-Biosciences), or IL-1β (Invitrogen) enzyme-linked immunosorbent assay (ELISA) kits. Cytokine production was measured with technical duplicates in each of several biological replicates.