Figure 2. Temperature-dependent binding and internalization of a2NTD by monocytes. (A–D) Peripheral blood mononuclear cells (PBMCs) were pre-incubated at 4°C or 37°C for 30 min, followed by the addition of 10 μg/mL a2NTD conjugated to Alexa Fluor 488 (a2NTD-AF488) for 1 h and staining with an anti-CD14 PE-Texas red antibody for 30 min. Some of the samples were treated with 0.1% trypsin after a2NTD-AF488 incubation to remove surface bound a2NTD. Cells were then placed on slides using a cytospin and imaged using a Nikon fluorescent microscope. Representative images from three independent experiments are shown: PBMCs incubated at 4°C, followed (A) or not (B) by trypsin treatment; PBMC incubated at 37°C, followed (C) or not (D) by trypsin treatment.