Preexisting sCD40L decreased IL-12 production from monocytes after in vitro stimulation. (A) Monocytes were isolated from PBMCs of a healthy donor using CD14 beads. The cells (10⁶/mL) were incubated with 2 μg/mL of sCD40L for 20 hours, after which IFN-γ (300 U/mL) and Golgi Stop (0.8 μL/mL) were added. Two hours later, a second signal (50 ng/mL LPS) was added to the culture. Cells were harvested 6 hours after the initial IFN-γ stimulation, and intracellular staining of IL-12 was performed. (B) Summary of data from 5 individual assays described in panel A of monocytes derived from 5 PBMC samples. The data were repeated 4 times. Statistical analysis was performed using a paired t test. SSC indicates side scatter.