Related to Fig. 1 . (A) Flow cytometry analysis of cell viability and infection rate of uninfected Jurkat cells (NI) or Jurkat cells infected with NLAD8-VSVG for 2 d (HIV-2d) or 7 d (HIV-7d). Left: Representative kinetics of Jurkat cell viability (%, red) and infection rate (% of HIV-p24+, green); frames indicate the time post-infection (2 d, yellow; 7 d, brown) selected for the rest of the study. Middle: Representative dot plot of Annexin V and LIVE/DEAD signals and gating strategy for selection of dead cells (LIVE/DEAD positive). Right: Quantification of % of dead cells (n = 6 donors, median ± interquartile range). (B) Flow cytometry analysis of 2 d-infected Jurkat cell viability in infected cells versus non-infected bystander cells. Left: Representative dot plot of HIV-p24 staining and gating strategy for selection of bystander and infected cells. Middle: Representative dot plots of LIVE/DEAD signals and gating strategy for selection of dead cells. Right: Quantification of % of dead cells in bystander and infected cells (n = 9 donors, mean ± SD). (C–F) Analysis of the three mechanisms of HIV-1 transfer from infected Jurkat cells to MDMs. (C and D) MDMs loaded with LysoTracker (red) were co-cultured with Jurkat cells infected with HIV-1 for 2 or 7 d and pre-loaded with CellTracker (gray). (C) Representative images of MDMs infected by phagocytosis (top), cell fusion (middle), or a synapse-like mechanism (bottom); HIV-p24 in green and nuclei in blue (DRAQ5). Infected MDMs were delineated by dotted lines. Scale bars, 20 µm. (D) Fluorescence intensity of HIV-p24 (green), LysoTracker (red), and CellTracker (black) signals along the dashed straight lines from right panels in C. (E) MDMs were co-cultured with Jurkat cells infected with HIV-1 (for 2 or 7 d) and pre-loaded with Celltracker (blue) and pHrodo (red). Representative images of MDMs infected by cell fusion (top) or phagocytosis (bottom). HIV-p24 in green and F-actin (phalloidin) in gray. Scale bars, 10 µm. (F) Quantification of the number of nuclei (DRAQ5+) in HIV-p24 + MDMs, from at least 200 cells per conditions. Quantification of a representative experiment is shown, from three experiments. (E) Flow cytometry analysis of MDMs co-cultured with uninfected (NI) or 2 d-infected Jurkat cells (HIV). Left: Representative dot plots of HIV-p24 and CD3 stainings and gating strategy to determine the % of CD3⁺ among HIV-p24 + MDMs. Right: Quantification. Statistical analyses: (A) Kruskal–Wallis multiple comparison test and (B) Paired t test. * P ≤ 0.05; ** P ≤ 0.01.