Chapter 3 54 methodology, after validation of markers of interest, will be useful for EV analysis in many different sample types and in a plethora of clinical settings. # Criteria Reasoning 1 Standardization of SSC signals Allows estimation of particle sizes 2 Single Spot Fluorescence Single particle analysis / no coincidence events 3 Colocalization of fluorophores Indicating the presence of markers in the same particle/event 4 Signal disappears after Detergent Lysis Confirmation that detected events are of biological origin 5 Linear correlation with Dilution factor Single particle analysis & confirmation that events are biological Table 1 - Criteria for events to be classified as true single EV by IFCM. MATERIALS & METHODS Processing and storage of human blood plasma (Steps I – III) The collection and processing of samples from 5 healthy human individuals (2 males, 3 females, average age: 43.4 years, age range: 31-56 years) was approved by the Medical Ethical Review Board (MERB number MEC-2018-1623) and conducted in accordance with the Declaration of Helsinki. All individuals provided written informed consent. In brief, 12 mL of blood was collected (one drawing) from each individual into two BD Vacutainer® K3-EDTA-coated collection tubes (BD Biosciences, San Jose, USA) (Figure 9 – step I). Whole blood was centrifuged (Heraeus Multifuge 1S) at 1910 x g for 10 minutes at room temperature (Figure). The plasma layer was then collected - leaving ~1 mm of plasma above the buffy coat - and centrifuged (Heraeus Fresco) at 16,000 x g for 10 minutes at room temperature in 1mL aliquots using Safe-Lock Eppendorf tubes (Eppendorf AG, Hamburg, Germany). The resulting platelet-poor plasma (PPP) was first pooled before being divided into 700-µL aliquots in cryovials containing 28 µL of a 25x concentrated protease inhibitor cocktail solution (4% v/v) (cOmplete Protease inhibitor cocktail tablets, Roche, Mannheim, Germany) according to the manufacturers’ instructions and stored at -80 °C (Figure 9 – step III).
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