PE Mouse Anti-Human CD31 Clone L133.1

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	Plasma samples were thawed at 37o C and centrifuged at 17,000 g for 10 min at RT. Pelleted material was resuspended in AnnV-binding buffer according to manufacturer´s instructions and aliquoted into BD Truecount Tubes

Protocol tips
Plasma samples were thawed at 37o C and centrifuged at 17,000 g for 10 min at RT. Pelleted material was resuspended in AnnV-binding buffer according to manufacturer´s instructions and aliquoted into BD Truecount Tubes

Publication protocol

Effects of hypobaric hypoxia at high altitude on extracellular vesicles (EV) in plasma were analyzed by flow cytometry (Fig 1A). This is currently the most widely used and accepted methodology for EV analyses [12]. In recent years, EV attracted much academic as well as clinical attention due to their assumed function as signaling units and biomarkers in a plethora of physiological and pathophysiological contexts [13, 14]. Large consortia strive to assess and establish protocols for standardization and quality control of qualitative as well as quantitative characterization of EV [12–14] as prerequisites for reliable routine application. As previously described (17) we here followed basic, and well-established protocols for flow cytometrical analysis of EV, [15–17]. EV were identified by size and granularity, and further specified by phosphatidylserine (PS) surface exposure (AnnexinV-binding). Platelet-derived (P)EV and endothelial cell-derived (E)EV were defined by platelet endothelial cell adhesion molecule (PECAM-1, CD31) surface antigen expression. The EV origin was further differentiated by platelet glycoprotein Ib alpha chain (GPIb, CD42b) expression on PEV (CD42b-positive) versus EEV (CD42b-negative). Flow cytometry of surface expression of CD31 (PECAM-1) and CD42b (GPIb, CD42b) on EV is a widely used method to discriminate PEV (CD31pos CD42bpos) from EEV (CD31pos CD42bneg)[15]. Plasma EV were analyzed as published previously [16]. Briefly, plasma samples were thawed at 37o C and centrifuged at 17,000 g for 10 min at RT. Pelleted material was resuspended in AnnV-binding buffer according to manufacturer´s instructions and aliquoted into BD Truecount Tubes (all BD Biosciences, Heidelberg, Germany). Samples were incubated with APC anti human CD42b and PE anti human CD31 in the dark at RT for 15 min before analysis with a FacsCalibur (all BD Biosciences). For analysis, gating started with total plasma EV, including the Truecount microbeads for quantification of EV/ml (Fig 1A, left), before AnnVpos EV (Fig 1A, middle) were further analyzed for CD31 and CD42 expression in order to differentiate between CD31pos CD42bpos PEV and CD31pos CD42bneg EEV (Fig 1A, right).

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