An Imaging Flow Cytometry-Based Methodology for the Analysis of Single Extracellular Vesicles 3 67 Framework Criteria What to report Please complete each criterion 2.1 Sample staining details State any steps relating to the staining of samples. Along with the method used for staining, provide relevant reagent descriptions as listed in MIFlowCyt guidelines (Section 2.4 Fluorescence Reagent(s) Descriptions). mAbs used: The monoclonal antibodies (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,k-APC, clone MOPC-21 (200 µg/mL, BioLegend); and IgG2a,k-APC, clone RTK2758 (200 µg/mL, BioLegend). mAb preperation: All mAbs were centrifuged for 10 minutes at 16,000 x g to reduce the number of (potential) mAb clumps. 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. mAb pre-dilutions All tetraspanin mAbs were diluted 30-fold in fPBS before staining (Final concentrations: CD9: 0.2 µg/mL, CD63: 6.6 µg/mL, CD81: 6.6 µg/mL); CD31-BV421 (anti-human) and CD31-APC (anti-mouse) were diluted 1000-fold (Final concentration: 50 ng/mL) and 62.5-fold (Final concentration: 3.2 µg/mL), respectively. The anti-tetraspanin antibody mixture was made by combining anti-CD9/anti-CD63/anti-CD81 in the same stock solution. CFDA-SE Stock solution preperation: A carboxyfluorescein diacetate succinimidyl ester (CFDA-SE) stock solution was made with the Vybrant™ CFDA-SE Cell Tracer Kit from Invitrogen immediately prior to use according to the manufacturer’s instructions: CFDA-SE powder was spun down using a table-top centrifuge, and 18 µL of dimethylsulfoxide (DMSO) was added. The mixture was thoroughly resuspended and incubated at room temperature for 10 – 15 minutes in the dark. The dissolved CFDA-SE was added to a total volume of 1.782 mL of fPBS to create a 50 µM CFDA-SE stock solution. Similar to the protocol used to prepare mAbs, this stock solution was centrifuged for 10 minutes at 16,000 x g to reduce potential CFDA-SE clumps; the top layer was carefully harvested before being added to the samples.
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