Facs diva data acquisition and analysis software

Size determination of MVs_MUC1 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 MVs_MUC1 was evaluated by flow cytometry. MVs_MUC1 (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), MVs_MUC1 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).

DC phenotype was analyzed by flow cytometry. Briefly, DCs were harvested, washed in phosphate-buffered saline (PBS) and resuspended at 10⁶ cells/ml. Each experimental sample was of 2 × 10⁵ cells/tube (BD Biosciences, NJ, USA), cells were incubated with specific fluorochrome-conjugated antibodies for 30 min at 4°C in the dark. After washing in PBS (two times), the cells were analyzed. The following monoclonal antibodies (mAb) were used: anti-HLAII-DR-APCH7 and anti-CD86-Pecy7, from BD Biosciences; anti-CD14-BB700, anti-CCR7-AlexaFlour647, anti-CD83-Pe, anti-CD40-BB515, anti-ICOS-L-Pe from BioLegend (San Diego, CA); and anti-VEGFR-1-PE from R&D Systems. MoAbs anti-IgG₁-BB515, -PE, -Pecy7, and -BB700; -AlexaFluor647; and -APC-H7 (BioLegend) were used as isotype controls. All mAbs were purchased from BD Biosciences and BioLegend. Flow cytometry analysis was performed using FACSCanto II flow cytometer running FACS Diva data acquisition and analysis software (BD Biosciences).