Figure 4. Binding of a2NTD and intracellular cytokine production in monocyte subpopulations. (A–D) Peripheral blood mononuclear cells (PBMCs) were incubated with 10 μg/mL a2NTD conjugated to Alexa Fluor 488 (a2NTD-AF488) for 1 h and then surface stained with anti-CD14-Pacific Blue and anti-CD16-PE antibodies. (A) Percentage of monocyte populations positive for a2NTD-AF488. (means ± SEM, n = 10, # p < 0.005, CD14²⁺CD16⁺ vs. CD14²⁺CD16^- or CD14⁺CD16⁺cells). (B–D) PBMCs were kept in control conditions or stimulated with 10 ng/mL a2NTD for 18 h and sequentially stained for surface expression of CD14/CD16 and the intracellular expression of interleukin(IL)-1α: (B), IL-1β (C) and tumor necrosis factor α (TNFα). (D) The bar graph reports the percentage of a2NTD-positive cells. (means ± SEM, n = 5,, *p < 0.05, ** p < 0.001, compared with unstimulated cells of the same subpopulation).