Figure 10.

Conditioned medium of cancer cells promoted PMA-induced NETs in human neutrophils. Human neutrophils were treated with a vehicle (Control), PMA (100 nM) or PMA (100 nM) in combination with HMGB1-neutralizing IgG (5 μg/mL) for 3 h. For the study of cancer cell effects, supernatants of subconfluent A549 and T24 cells over a period of 24 h were collected as Conditioned Medium (CM). Human neutrophils were treated with A549 CM (with an equal volume of fresh medium), T24 CM (with an equal volume of fresh medium), or in combinations as indicated for 3 h. For the neutralization study, A549 CM and T24 CM were incubated with HMGB1-neutralizing IgG (5 μg/mL) for 15 min prior to being added to the neutrophils. The treated neutrophils were subjected to flowcytometric analysis for the measurement of citrullinated histone H3 positivity with an indicated primary antibody and an Alexa Fluor 488-labeled secondary antibody. Representative plots and quantitative data are shown. * p < 0.05 vs. untreated group, # p < 0.05 vs. PMA group, % p < 0.05 vs. PMA/A549 CM group, and & p < 0.05 vs. PMA/T24 CM group, n = 3.