Anti hla dr bv570

Co-cultures were harvested 36 h after initiation, treated with 20 μg/ml of human IgG and stained with anti-HLA-DR BV570 (Clone L243, Biolegend) for 30 min at 4°C. Cells were then fixed with 2% methanol-free formaldehyde for 30 min, washed, and stained with phalloidin AF-647 in 0.3% saponin for 30 min at 4°C. At least 50,000 events were acquired for each sample and analyzed using the IDEAS Software version 5.0. To evaluate the HIV migration to the IS, we first quantified the iGFP-HIV intensity at the synapse divided by the total iGFP-HIV intensity. Second, we quantified the actin intensity at the interface divided by the total actin staining. Third, we plotted these two features to track the co-localization of HIV with polymerized actin at the IS. In some experiments, DCs were treated with Zidovudine (AZT, 1 μM) at the time of HIV infection to confirm HIV infection. In addition, cells were treated with Cytochalasin D (20 μM) or Nocodazole (100 μM) at the time of co-culture, as control of actin polymerization and viral movement.