*2.6. Flow Virometry*

Flow virometry experiments were performed with a CytoFLEX LX (Beckman Coulter) with violet side scatter (V-SSC) 405 nm filter configuration at Servei de Cultius Cel·lulars, Producció d'Anticossos i Citometria (UAB, Barcelona, Spain). The different steps required in the analysis are described in Figure 6A. Measurements from different experiments were standardized using a mixture of Megamix-Plus Side Scatter and Forward Scatter (FSC) fluorescent polystyrene beads (100, 160, 200, 240, 300, 500 and 900 nm; Biocytex, Marseille, France). The threshold of height trigger signal in Violet Side Scatter (V-SSC) was manually adjusted to 1200 and laser gains were set as 72, 9 and 106 for FSC, V-SSC and B525-FITC, respectively. Samples were diluted with 1 X Dulbecco's phosphate-buffered saline (DPBS, Thermo Fisher Scientific) until the abort rate value was below the 2%. Three-hundred thousand events were analyzed per sample at a flow rate of 10 μL/min. V-SSC vs B525-FITC density plots were used to gate the different EVs and VLPs populations. Gating was manually adjusted for each channel. Results were analyzed with the CytExpert v.2.3 software (Beckman Coulter). Nanoparticle concentrations were calculated with Equation (1):

$$\text{Particle concentration} \left(\frac{\text{Eventts}}{\text{mL}}\right) = (\text{Eventts}) \cdot \frac{\mu\text{L}}{\text{mL}} \cdot \text{Dilution} \tag{1}$$

Particle size diameters based on the median violet side scatter were calculated by Mie correlation [43] using FCMPASS software [33]. Megamix-Plus SSC and FSC fluorescent polystyrene beads were used as the reference material with a refractive index (RI) of 1.633 with a 405 nm illumination wavelength [43]. The following RI were used for Mie correlation: 1.374 (vesicle cytosol), 1.394 (vesicle upper cytosol), 1.354 (vesicle lower cytosol), 1.474 (vesicle membrane) and 1.345 (vesicle surrounding medium) considering 405 nm as the illumination wavelength. The vesicle membrane thickness was defined as 10 nm [43]. The half angle parameter was set as circular aperture geometry. The maximum diameter was fixed as 3000 nm, the triggering threshold as 60 arbitrary units (a.u.) and the log decade number parameter comprised between 0–6. Thereafter, HIV-1 Gag-eGFP VLP and EV mean sizes were exported with the FlowJo v.10 software (BD Biosciences).
