An Imaging Flow Cytometry-Based Methodology for the Analysis of Single Extracellular Vesicles 3 55 Processing and storage of mouse blood plasma All the procedures and animal housing conditions were carried out in strict accordance with current EU legislation on animal experimentation and were approved by the Institutional Committee for Animal Research (DEC protocol EMC No. AVD101002016635). Six weeks male C57BL/6J (JAX,GSP) mice (Jackson Labs, Bar Harbor, ME) were housed in Erasmus MC animal facility and housed in groups of 2-3/cage. They were maintained on a 12:12 h light-dark cycle and allowed ad libitum access to water and standard rodent food. The mice were anesthetized and blood (approximately 0.8 mL) was collected via the left ventricle using a 23-25 gauge needle. To ensure euthanasia of the animal post-procedure, mice were killed by cervical dislocation. Antibody preparation (Step IV) All monoclonal antibodies (mAbs) were centrifuged for 10 minutes at 16,000 x g to reduce the number of (potential) mAb clumps (Figure 9 – step IV). A volume of the top layer of each centrifuged mAb solution was carefully harvested (according to the dilutions needed, described below) and diluted in 0.22 µm-filtered PBS (fPBS) before being added to the samples (Figure 9 – step VI). The sample staining protocol is described under step VI. The mAbs used to stain human PPP were anti-CD9–APC, clone HI9a (6 µg/mL, BioLegend, San Diego, USA); anti-CD63–APC, clone H5C6 (200 µg/mL, BioLegend); and anti-CD81–APC, clone 5A6 (200 µg/mL, BioLegend. Human and mouse PPP were both stained with anti-human CD31–BV421, clone WM-59 (50 µg/mL, BioLegend) and anti-mouse CD31-APC, clone 390 (200 µg/mL, BioLegend). Isotype controls used were IgG1,k-BV421, clone MOPC-21 (100 µg/mL, BioLegend); IgG1,kAPC, clone MOPC-21 (200 µg/mL, BioLegend); and IgG2a,k-APC, clone RTK2758 (200 µg/mL, BioLegend). Optimal mAb concentrations were determined by performing separate titration experiments for each mAb on human PPP and fPBS samples in parallel. The optimal concentration of each mAb was defined as the concentration that yielded the best discrimination between sample (PPP) and background (fPBS). All tetraspanin mAbs were diluted 30-fold in fPBS before staining (Final concentrations: CD9: 200 ng/mL, CD63: 6.6 µg/mL, CD81: 6.6 µg/mL); CD31-BV421 (anti-human) and CD31-APC (antimouse) were diluted 1000-fold (Final concentration: 50 ng/mL) and 62.5-fold (Final
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