Anti mouse cd62p fitc

Mouse whole blood was treated with either 10 μM ADP or 1.0x10⁵ pg p24 EcoHIV for 30 min, 1 hr, and 2 hrs at 37°C. Subsequently, blood was processed and analyzed for platelet activation by flow cytometry. Briefly, whole blood was fixed with 4% paraformaldehyde and washed twice with 1 ml staining buffer (1x PBS containing 2% BSA). Red blood cells (RBCs) were then lysed using ACK lysis buffer (Life Technologies, Grand Island, NY) and the remaining cells were washed twice in staining buffer. Cells were stained with 2.5 μl anti-mouse CD61-PE (AbD Serotec, Oxford, U.K.) and 1 μl anti-mouse CD62P-FITC (BD Biosciences, San Jose, CA). Following the staining, samples were acquired using a flow cytometer (Accuri C6; Accuri Cytometers, Ann Arbor, MI). Platelets were gated based on forward and side scatter, followed by assessment of the platelet activation marker CD62P. Sizing beads (Mega Mix; BioCytex, Marseille, France) were used to delineate the platelet gate (0.9–3 μm). Unstained cells and cells stained with only CD61 were used as controls. 20,000 events were acquired to measure platelet percentages and activation in terms of CD62P expression.

Platelet activation was assessed in human whole blood via CD62P (P-selectin) surface expression, using a previously described method [11 (link), 13 (link)], within 1h of the blood draw. Similarly, platelet activation in mouse blood was assessed using 2.5μL anti-mouse CD61-PE (catalog # 561910) and 1μL anti-mouse CD62P-FITC (catalog # 561923; BD Biosciences, San Jose, CA).