For calibration of the observation spot sizes using STED-FCS, SLBs were prepared, as previously described (61), by spin-coating a coverslip with a solution of DOPC (1 mg/ml) and KK114-DPPE (0.5 μg/ml) in CHCl3/MeOH. Coverslips were cleaned by piranha solution (3:1 sulfuric acid and hydrogen peroxide). The lipid bilayer was formed by rehydrating with SLB buffer containing 10 mM Hepes and 150 mM NaCl (pH 7.4). Calibration of the confocal observation spot size was made with a 50 nM solution of rhodamine in pure water using D = 280 μm2/s as the diffusion coefficient.

For lipid mobility measurements inside and outside Gag assembly sites in mammalian cells, 3 × 105 Jurkat T cells 72 hours after infection with NL4.3 HIV-1 Gag-iGFP virus particles or 24 hours after transfection with NL4.3 HIV-1 Gag-eGFP–expressing plasmid were resuspended in 200 μl of Leibovitz’s L-15 medium (Sigma-Aldrich) and incubated with 25 nM of the indicated fluorescent lipid analog probe for 5 min at 37°C. Cells were then washed three times in suspension in L-15 medium, resuspended in 250 μl of L-15 medium, and adhered to solution poly-l-lysine (0.1 mg/ml) (Sigma-Aldrich)–coated glass surface of eight-well Ibidi μ-Slides (Ibidi, Martinsried, Germany) at 37°C 30 min prior to microscopy measurements.

For lipid mobility measurements on myr(−) Gag containing SLBs, prior to experiments, citrate buffer was changed to protein buffer [10 mM Hepes (pH 7.4), 150 mM KCl, and 2 mM EDTA], followed by injection of 1 μM myr(−) Gag.

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