An Imaging Flow Cytometry-Based Methodology for the Analysis of Single Extracellular Vesicles 3 77 Requirement Please Include Requested Information 2.1.1.2. Biological sample source description See above 2.1.1.3. Biological sample source organism description Healthy human individuals – 2 male, 3 female, age range 31 – 56 (mean 43,4). Mouse – see above. 2.1.2.2. Environmental sample location NA 2.3. Sample treatment description Bloods were drawn, processed and stored as described above. For staining, 30 uL of PPP was added to a pre-defined volume of fPBS (dependant on the volume of mAb staining - total volume after mAb addition was set at 130 µL): 12.5 uL of the stock solutions containing mAbs labelled with –APC and 5 µL of the stock solutions containing mAbs labeled with –BV421 were added, resulting in the following concentrations used per test: anti-CD9 – 2.5 ng, anti-CD63 – 83 ng, anti-CD81 – 83 ng, anti-CD31 (anti-human) – 1 ng, anti-CD31 (antimouse) – 40 ng per test. Equivalent amounts of isotype control was used for each antibody. Samples were then incubated overnight at 4 °C to ensure optimal saturation of the available EV epitopes; this incubation time was determined by adding the anti-tetraspanin antibody mix to fPBS and PPP samples and performing acquisition at set intervals (1/3/6 hours and O/N). CFDA-SE labeling was performed on the day of data acquisition by adding 100 µL of the 50 µM CFDA-SE stock solution to the samples, followed by 30 minutes of incubation at room temperature in the dark. Control samples not stained with CFDA-SE were incubated with 100 µL fPBS instead. All samples were brought to a total volume of 380 µL using fPBS before IFCM measurements. 2.4. Fluorescence reagent(s) description 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 antiCD81–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). 3.1. Instrument manufacturer LUMINEX 3.2. Instrument model ImageStreamX MkII
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