Colocalization of HIT ICs, FcRn, and FcγRIIa on THP-1 cells upon treatment with IgG-containing ICs. (A) THP-1 cells immobilized on RetroNectin were left untreated (top) or incubated with PF4 + KKO complexes (bottom) for 15 minutes as in Figure 1A, washed, and fixed in 4% paraformaldehyde in PBS. (B) Inhibition of HIT ICs binding and coclustering with FcRn and FcγRIIa by α-FcRn and α-FcγRIIa mAbs on the surface of THP-1 cells. THP-1 cells were preincubated with control human IgG4 and control mouse IgG (TRA; 200 μg/mL both; top), α-FcRn antibody (Ab; 200 μg/mL of SYNT001; second row) or with α-FcγRIIa Ab (200 μg/mL IV.3; third row) or with both Abs (bottom) for 30 minutes before addition of PF4 and KKO ICs for 15 minutes. The cells were then washed, fixed, and stained. PF4/KKO ICs, FcRn, and FcγRIIa were detected using Alexa-568–conjugated α-PF4/heparin mAb KKO (red), Alexa-488–conjugated α-human FcRn mAbs (green), and Alexa-647–conjugated FcγRIIa mAbs (pseudocolored in white). Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI; blue). Individual red, green, and deep-red channels and their overlays with blue channel (nuclei staining; overlay + DAPI) are shown. Staining with negative control Abs is shown within the insets in each panel. Scale bars, 10 μm.