Chapter 3 76 Requirement Please Include Requested Information 1.1. Purpose To develop a protocol for the direct measurement of Extracellular Vesicles (EV) in unprocessed (human) plasma samples. 1.2. Keywords Unprocessed Human Plasma; Extracellular Vesicles; Imaging Flow Cytometry; Quantify; Phenotype; Diagnostic Platform 1.3. Experiment variables Platelet-poor plasma (PPP) samples from 5 healthy individuals and/or six week old male C57BL/6J (JAX,GSP) mice (Jackson Labs, Bar Harbor, ME) were stained with CFDA-SE, anti-tetraspanin antibodies (CD9, CD63, CD81) and CD31, and measured with Imaging Flow Cytometry (IFCM). 1.4. Organization name and address Erasmus Medical Center, University Medical Center Rotterdam, The Netherlands. Wytemaweg 80, 3015 CN, Rotterdam 1.5. Primary contact name and email address Wouter W. Woud, wouterwwoud@gmail.com 1.6. Date or time period of experiment 2020 - 2021 1.7. Conclusions Imaging Flow Cytometry (IFCM) can be used to identify, quantify and phenotype fluorescently tagged EV ≤240 nm in unprocessed (human) plasma samples. 1.8. Quality control measures The instrument calibration tool ASSIST® was used upon each startup to optimize performance and consistency between experiments. Additionally, commercially available mixtures of FITC-fluorescent polystyrene beads of known sizes (Megamix-Plus FSC – 900, 500, 300 and 100 nm, and Megamix-Plus SSC – 500, 240, 200, 160 nm), as well as Rainbow Calibration Particles (RCP-05-5, lot AL01, Spherotech), were used in calibrating and standardization of the IFCM platform. 2.1.1.1. (2.1.2.1., 2.1.3.1.) Sample description Platelet-poor plasma (PPP) obtained from 5 healthy individuals was used in this study. From each of the 5 healthy individuals, 12 mL of blood was collected (one drawing) into two BD Vacutainer® K3-EDTAcoated collection tubes (BD Biosciences, San Jose, USA). Whole blood was centrifuged (Heraeus Multifuge 1S) at 1910 x g for 10 minutes at room temperature. 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. The resulting PPP was 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. Additionally, PPP was generated from mice. 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.
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