An Imaging Flow Cytometry-Based Methodology for the Analysis of Single Extracellular Vesicles 3 61 Figure 9 – Schematic overview of the sample processing and staining protocol. EDTA whole blood (I) is centrifuged for 10 minutes at 1910 x g, after which the plasma top layer (II) is harvested and subjected to centrifugation for 10 minutes at 16,000 x g. The resulting plateletpoor plasma (PPP) is collected and stored as aliquots in cryovials containing an anti-protease inhibitor stock solution (4% v/v) at -80 °C (III). Prior to use, monoclonal antibodies and CFDA-SE stock solutions are centrifuged for 10 minutes at 16,000 x g to reduce the number of potential clumps of fluorescent particles (IV & V). mAbs are added to 30 µL of PPP, and samples are brought to a total volume of 130 µL using filtered PBS (VI) and incubated overnight (O/N) at 4 °C in the dark. CFDA-SE is added on the day of acquisition and incubated for 30 minutes at room temperature in the dark. Samples are brought to a total volume of 380 µL using filtered PBS and are assessed using a Luminex ISx MKII imaging flow cytometer (VII). After initial acquisition, detergent treatment is performed for each sample by adding 20 µL 10% (v/v) TritonX-100 solution followed by 30 minutes incubation at room temperature. Permission to use the image of the Imagestream (ISx) Imaging Flow cytometer (VII) was granted by Luminex.
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