Size determination of MVsMUC1 was performed by Nanoparticle Tracking Analysis (NTA) technology (30 (link)). MVs were thawed on ice and diluted in PBS between 1:500 and 1:20,000 to achieve the optimal number of MVs/mL. Three videos (30 s each) were recorded for each sample loading, employing the NanoSight NS300 instrument (Malvern Instruments Ltd, Malvern, UK). Measurements were performed employing the NTA 2.3 analytical software. Results were shown as the average of the three recordings.
MUC1 expression on MVsMUC1 was evaluated by flow cytometry. MVsMUC1 (5 μg/sample) were incubated with the anti-MUC1 MoAb Ma552 (Monosan) (1:100 for 30 min, 50 μL/sample, RT). After washing in PBS w/o Mg++ and Ca++(1 mL/sample, 30 min at 13,000 rpm, RT), MVsMUC1 were incubated with FITC-conjugated anti-mouse antibody (1:600; Jackson-Immunoresearch Laboratories, 50 μL/sample). MoAb MOPC21 (1:100; Sigma-Aldrich) was employed as isotype control. To exclude background noise, flow cytometry analysis was performed setting the lowest Forward Scatter Threshold [300] and the highest FSC/SSC voltage. A total of 30,000 events were acquired with low flow rate, using a FACSCanto II flow cytometer running FACSDiva data acquisition and analysis software (Becton Dickinson).
MUC1 expression on MVsMUC1 was evaluated by flow cytometry. MVsMUC1 (5 μg/sample) were incubated with the anti-MUC1 MoAb Ma552 (Monosan) (1:100 for 30 min, 50 μL/sample, RT). After washing in PBS w/o Mg++ and Ca++(1 mL/sample, 30 min at 13,000 rpm, RT), MVsMUC1 were incubated with FITC-conjugated anti-mouse antibody (1:600; Jackson-Immunoresearch Laboratories, 50 μL/sample). MoAb MOPC21 (1:100; Sigma-Aldrich) was employed as isotype control. To exclude background noise, flow cytometry analysis was performed setting the lowest Forward Scatter Threshold [300] and the highest FSC/SSC voltage. A total of 30,000 events were acquired with low flow rate, using a FACSCanto II flow cytometer running FACSDiva data acquisition and analysis software (Becton Dickinson).
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