In most of the experiments, EVs were labeled with the fluorescent membrane dye DiR or PKH67 (see above) to distinguish EVs from background noise. MC38-EVs were blocked with rat anti-mouse FcγIII/II receptor (CD16/CD32) blocking antibodies (2 μg/ml) for 15 min at 4°C and stained with mouse anti-HER2-AF647 (1:50, clone 24D2, BioLegend) for 1h at 4°C. Between each step, EVs were washed using Vivaspin® 500 columns (300,000 MW cutoff, Sartorius), as described above.
EV samples were typically diluted in PBS at 50-100 μg/ml, and analyzed using an Attune NxT apparatus (Life Technologies). Acquisition settings were optimized for detection of EV populations carrying green, red or near-infrared fluorescence, or a combination thereof. The conventional blue side scatter (SSC, 488 nm) was replaced by the violet side scatter (vSSC, 405 nm) and the acquisition threshold was set at 0.2 on the vSSC. EVs were gated based on their positivity for DiR and vSSC properties. Compensation was performed using single-stained OneComp eBeads (Thermo Fisher Scientific).
EV samples were typically diluted in PBS at 50-100 μg/ml, and analyzed using an Attune NxT apparatus (Life Technologies). Acquisition settings were optimized for detection of EV populations carrying green, red or near-infrared fluorescence, or a combination thereof. The conventional blue side scatter (SSC, 488 nm) was replaced by the violet side scatter (vSSC, 405 nm) and the acquisition threshold was set at 0.2 on the vSSC. EVs were gated based on their positivity for DiR and vSSC properties. Compensation was performed using single-stained OneComp eBeads (Thermo Fisher Scientific).