Pe mouse anti human cd61

The generation of PDMPs was adapted from Recabarren-Leiva et al [24] (link). Briefly, 400 μL of platelets (PRP or washed) were dispensed in siliconized glass cuvettes at 37 °C with constant stirring at 1000 r.p.m. preincubated for 15 minutes with LPS (0–9 μg/mL) and stimulated with ADP (1 or 4 μM) for 30 and 60 minutes. Then centrifuged at 16,000 g for 60 minutes at 4 °C and the supernatants were analyzed on Accuri™ C6 flow cytometer (BD Biosciences, San Jose, CA).
We identified the PDMPs according to two parameters, the first based on size, complexity (Forward scatter/side scatter; FSC/SSC) and then their positivity against specific markers. Thus the samples were incubated with 5 μL of PE Mouse Anti-Human CD61 (PE, Phycoerythrin and CD61 is a transmembrane glycoprotein that is also known as platelet glycoprotein IIIa; BD Pharmingen) for 30 minutes in the dark. Also un-labelled samples as autofluorescence control were used.

Human CD45 microbeads were obtained from Miltenyi Biotec (Bergisch Gladbach, Germany). Prostaglandin E₁ (PGE₁), and JC-1 were purchased from Sigma-Aldrich (St. Louis, MO, USA). FITC Annexin V Apoptosis Detection Kit and PE Mouse Anti-Human CD61 were obtained from BD Biosciences (Heidelberg, Germany). VerifyNow P2Y12 assay was purchased from Accumetrics (San Diego, CA, USA), and adenosine diphosphate (ADP) was from Helena Laboratories (Beaumont, TX, USA). Anti-Bcl-2, anti-Caspase-3, and anti-β-actin rabbit antibody were sourced from Cell Signaling Technology (Danvers, MA, USA). ABT-737 was from Selleck Chemical (Houston, TX, USA). mirVana miRNA Isolation Kit, Taqman microRNA primers (hsa-miR-145-5p and hsa-miR-15b-5p), Taqman microRNA Reverse Transcription Kit, Taqman Universal Master Mix II, mirVana miRNA mimic and inhibitor (hsa-miR-15b-5p), mirVana miRNA mimic and inhibitor negative control #1, and Lipofectamine RNAiMAX reagents were purchased from Applied Biosystems (Foster City, CA, USA).