Computational protocol: Differential Interaction of Platelet-Derived Extracellular Vesicles with Leukocyte Subsets in Human Whole Blood

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Protocol publication

[…] EVs were characterized in PFP, which was generated from freshly drawn whole blood as described above. Samples were diluted 1:400 in sterile-filtered PBS prior to analysis. Staining of EVs was performed for 15 min in the dark using FITC-conjugated lactadherin as marker for phosphatidylserine in combination with CD41-PC7 and CD235a-APC-AF750 as markers of platelet and red blood cell origin, respectively. All antibodies were centrifuged at 17,000 g for 10 min at 4 °C prior to use to remove precipitates. Flow cytometry was performed using a CytoFLEX flow cytometer (Beckman Coulter, Brea, CA) equipped with 405 nm, 488 nm, 561 nm, and 638 nm lasers. The triggering signal was set to the side scatter (SS) of the 405 nm laser and was combined with triggering on fluorescence. Fluorescent-green silica particles (1 µm; excitation/emission 485/510 nm; Kisker Biotech, Steinfurt, Germany) were used for calibration, and the EV gate was set below the 1 µm bead cloud as indicated in Supplementary Fig.  and . Samples were acquired for 2 min at a flow rate of 10 µl/min. To avoid carry-over effects, a 2 min washing step with sterile filtered double distilled water was performed between each measurement at a flow rate of 60 µl/min. Data were analyzed using the CytExpert Software Version 1.2 (Beckman Coulter). Buffer controls and fluorochrome labeled reagent controls are shown in Supplementary Fig. . The release of EVs during storage of whole blood and PFP was compared by analyzing EVs in freshly drawn PFP and after storage of PFP for 3 h at 37 °C with gentle agitation. Alternatively, PFP was obtained after storage of whole blood (3 h, 37 °C), and analyzed as described above, as shown in Fig.  and in Supplementary Fig.  (n = 3). [...] For flow cytometric characterization of classical, intermediate, and non-classical monocytes directly in whole blood, freshly drawn heparinized blood (200 µl) was stained with CD14-PE, CD16-PC5, CD66b-APC, and CD45-PB for 15 min and treated with 2 ml of lysis solution (IOTest 3, Beckman Coulter) for 10 min at RT to lyse red blood cells. The remaining cells were pelleted for 5 min at 150 g, washed with PBS, resuspended in 500 µl PBS, and analyzed on a Gallios flow cytometer (Beckman Coulter) equipped with 405 nm, 488 nm, and 638 nm lasers. Data were analyzed using the Kaluza Software (Beckman Coulter). For gating, platelets and remaining red blood cells were excluded based on their CD45 expression, granulocytes were excluded based on their CD66b expression, and lymphocytes were excluded based on their expression of CD45 and CD14. Monocyte subsets were identified based on their expression of CD14 and CD16 as classical monocytes (CD14++CD16−), intermediate monocytes (CD14++CD16+), and non-classical monocytes (CD14+CD16++) (n = 5). In addition to CD14 and CD16 surface expression, monocyte subsets were characterized via the exposure of chemokine receptors CCR2, CX3CR1, CCR5 as summarized in Fig. , (n = 3). […]

Pipeline specifications

Software tools CytExpert, Kaluza
Application Flow cytometry
Organisms Homo sapiens
Diseases Infection, Thrombosis