(A) Gating strategy for flow analysis of MNP complexes: A singlet gate was defined by plotting fluorescence height versus fluorescence width. The gate excludes events with a high width and high height that represent aggregated particles (for more details on gating, see Supplemental Figure 1. Supplemental material available online with this article; doi: 10.1172/JCI67042). (B and C) 2G12-coupled MNPs labeled with Alexa Fluor 488 were incubated with HIV-1_SF162 and stained with Alexa Fluor 647–labeled anti-gp120 antibody VRC01, and eFluor 450–labeled anti-CD45 antibody. CD45-positive events (B) are excluded from analysis, while gated CD45-negative MNP events positive for VRC01 (C) represent HIV-1 bound to the MNPs. (D) 2G12-coupled MNPs labeled with Alexa Fluor 488 were incubated with virus-free culture medium and stained with Alexa Fluor 647–labeled anti-gp120 antibody VRC01. Note the very low nonspecific binding of anti-gp120 antibodies to MNPs. (E) 2G12-coupled MNPs labeled with Alexa Fluor 350 were incubated with Δ-ENV-GFP HIV-1. Note the low binding of Δ-ENV-GFP HIV-1 to MNPs. (F–H) 2G12-coupled MNPs labeled with Alexa Fluor 488 were incubated with HIV-1_SF162 and stained with Alexa Fluor 647–labeled human IgG1 (Hu IgG1) (F) or eFluor 450–labeled IgG1 κ (G) isotype control antibodies for gp120 or CD45, respectively. Note the very low nonspecific binding of isotype control (ctrl) antibodies to HIV-1-MNP complexes. (H) 2G12-coupled MNPs labeled with Alexa Fluor 488 were incubated with a suspension of microvesicles produced by uninfected cells and stained with Alexa Fluor 647–labeled anti-gp120 antibody VRC01. Note the low binding of MNPs to microvesicles. On each plot, the fraction of events in their respective gate is expressed as a percentage of the total events in the plot.