Bead‐based multiplex flow cytometry assay

EVs isolated from Jurkat CCM (Fig 3B) and primary CD4⁺ T CCM (Fig EV2B) by SEC were subjected to bead‐based multiplex analysis by flow cytometry (MACSPlex Exosome Kit, human, Miltenyi). Samples were processed according to manufacturer's instructions, with 3 detection antibodies used separately. Particle counts quantified by NTA were used to estimate input EV amounts. 2 × 10⁹ EVs were diluted with MACSPlex buffer to a final volume of 120, and 10 µl of MACSPlex Exosome Capture Beads was added. Samples were incubated on an orbital shaker overnight at room temperature protected from light. After washing, detection antibodies (APC‐conjugated anti‐CD81 or anti‐CD63 [included in the kit] or 5 μl of anti‐CD3E [Miltenyi, 130‐113‐697]) were incubated for 1 h at RT. Flow cytometric analysis was performed with Aurora analyser (Cytek) and data analysed with FlowJo software (v10, FlowJo LLC). The 39 single bead populations were gated to allow determination of the APC signal intensity on the respective bead population and median fluorescence intensity (MFI) for each capture bead was measured. Out of the 37 markers analysed, only those detected in the proteome of Jurkat are shown.

100K pellets from control cells, SERINC3 KD cells and control cells after NL4‐3 EGFP‐Nef⁺ infection (Fig 6G) were analysed by the MACSPlex Exosome Kit as above, but with detection by a mix of APC‐conjugated anti‐CD9/CD81/CD63 antibodies provided by the manufacturer, and including a fixation step with 4% PFA for 1 h to inactivate the virus. 3–5 × 10⁸ fixed EVs and 15 µl of MACSPlex Exosome Capture Beads were used. Flow cytometric analysis was performed with a MACSQuant Analyzer 10 with the corresponding software (Miltenyi Biotec) following the acquisition recommendations for the MACSPlex Exosome kit (Miltenyi Biotec). FlowJo software (v10, FlowJo LLC) was used to analyse flow cytometric data. For each capture bead, background was corrected by subtracting respective MFI values from matched non‐EV controls that were treated exactly like EV‐containing samples, and values of the corresponding isotype control were further subtracted. Due to low amount of sample and fixation, some markers, such as CD3 and CD9, were not detected.