Chapter 3 66 Framework Criteria What to report Please complete each criterion 1.1 Preanalytical variables conforming to MISEV guidelines. Preanalytical variables relating to EV sample including source, collection, isolation, storage, and any others relevant and available in the performed study. From each of the 5 (human) healthy individuals , 12 mL of blood was collected (one drawing) into two BD Vacutainer® K3-EDTA-coated 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 platelet-poor plasma (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. 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 postprocedure, mice were killed by cervical dislocation. 1.2 Experimental design according to MIFlowCyt guidelines. EV-FC manuscripts should provide a brief description of the experimental aim, keywords, and variables for the performed FC experiment(s) using MIFlowCyt checklist criteria: 1.1, 1.2, and 1.3, respectively. Template found at www.evflowcytometry.org. 1.1 Aim: To develop an assay 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).
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